Anti-Laminin4 Antibodies Specific for LG4-5

ABSTRACT

The invention provides antibodies that specifically bind to the LG4-5 modules of the G domain of laminin α4. The antibodies can preferentially stain cancer or tumor cells or tissue. The antibodies can be used for detecting cancer, evaluating the efficacy of a cancer therapy, treating cancer, and treating obesity or obesity-related diseases, among other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 61/952,132, filed Mar. 12, 2014, U.S. Application No. 62/023,760, filed Jul. 11, 2014, and U.S. Application No. 62/068,349, filed Oct. 24, 2014, each of which is incorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file 455781SEQLIST.txt is 141 kilobytes, was created on Mar. 5, 2015, and is hereby incorporated by reference.

BACKGROUND

Cancer is a major public health problem. One in four deaths in the United States is due to cancer, and the lifetime probability of being diagnosed with an invasive cancer is approximately 40%. Siegel et al., CA Cancer J. Clin. 62:10-29 (2012). An estimated 500,000 Americans die from cancer each year (Siegel et al., CA Cancer J. Clin. 62:10-29 (2012)). Lack of specificity of conventional chemotherapies and consequent side effects limit the delivery of drug dosages needed to eliminate the majority of cancer cells.

Laminin proteins containing the laminin α4 chain have been reported to be expressed in some types of cancer. Syndecans, which are reported binding partners of laminin α4, have also been reported to be expressed in some types of cancer.

SUMMARY OF THE CLAIMED INVENTION

The invention provides antibodies that specifically binds to an epitope within the LG4-5 modules of the G domain of laminin α4. Some antibodies do not inhibit binding of laminin α4 to MCAM. Some antibodies bind to an epitope within LG4. Some antibodies bind to an epitope within LG5. Some antibodies bind to an epitope to which both LG4 and LG4 contribute residues. Some antibodies inhibit binding of laminin α4 to a heparan sulfate proteoglycan, such as syndecan-1, -2, -3, -4, a glypican, a betaglycan, CD44, perlecan, agrin, or collagen XVIII.

Some of the above antibodies selectively binds to a biological sample comprising cancer cells, such as melanoma cells, when compared to a control sample, optionally wherein the control sample and the biological sample comprise cells of the same tissue origin and optionally wherein binding of the antibody to the biological sample is at least 2-fold or 5-fold greater than the binding of the antibody to the control sample.

Some of the above antibodies compete for binding to laminin α4 with antibody 15F7 characterized by a mature heavy chain variable region of SEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17, or antibody 6C12 characterized by a mature heavy chain variable region of SEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27, or antibody 13G10 characterized by a mature heavy chain variable region of SEQ ID NOS:36 or 37 and mature light chain variable region of SEQ ID NO:38. Some of the above antibodies compete for binding to laminin α4 with antibody 15F7 characterized by a mature heavy chain variable region of SEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17, or antibody 6C12 characterized by a mature heavy chain variable region of SEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27 or 94, or antibody 13G10 characterized by a mature heavy chain variable region of SEQ ID NOS:36 or 37 and mature light chain variable region of SEQ ID NO:38.

Some of the above antibodies binds to the same epitope on laminin α4 as 15F7, 6C12, or 13G10.

Some of the above antibodies comprising three light chain CDRs and three heavy chain CDRs, wherein each CDR has at least 90% sequence identity to a corresponding CDR from the heavy and light chain variable regions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively). Some of the above antibodies comprising three light chain CDRs and three heavy chain CDRs, wherein each CDR has at least 90% sequence identity to a corresponding CDR from the heavy and light chain variable regions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27/94, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively).

Some of the above antibodies comprise three heavy chain CDRs and three light chain CDRs of 15F7, 6C12, or 13G10.

Some of the above antibodies are monoclonal. Some of the above antibodies are chimeric, humanized, veneered, or human. Some of the above antibodies have human IgG1 kappa isotype.

The invention further provides a humanized or chimeric 15F7 antibody that specifically binds to laminin α4, wherein 15F7 is a mouse antibody characterized by a mature heavy chain variable region of SEQ ID NO:16 and a mature light chain variable region of SEQ ID NO:17. Optionally the humanized antibody comprises a humanized heavy chain comprising three CDRs of the 15F7 heavy chain variable region (SEQ ID NO:16) and a humanized light chain comprising three CDRs of the 15F7 light chain variable region (SEQ ID NO:17). Optionally the humanized antibody comprises a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:58 and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:59. Optionally, the humanized antibody comprises a humanized heavy chain comprising three CDRs of the 15F7 heavy chain variable region (SEQ ID NO:16) and a humanized light chain comprising three CDRs of the 15F7 light chain variable region (SEQ ID NO:17). Optionally, any differences in CDRs of the mature heavy chain variable region and mature light chain variable region from SEQ ID NOS:16 and 17, respectively reside in positions H60-H65. In some humanized antibodies at least one of positions L42, L49, and L69 is occupied by N, S, and K, respectively, and at least one of positions H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 is occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively. In some humanized antibodies positions L42, L49, and L69 are occupied by N, S, and K, respectively, and H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 are occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively. In some humanized antibodies at least one of positions L8, L45, and L80 is occupied by S, R, and T, respectively. In some humanized antibodies at least one of positions L8, L45, L80, and L104 is occupied by S, R, T, and V, respectively. In some humanized antibodies at least one of positions H41 and H69 is occupied by A and L, respectively. In some humanized antibodies positions L8, L45, and L80 are occupied by S, R, and T, respectively. In some humanized antibodies position L104 is occupied by V. In some humanized antibodies positions H41 and H69 are occupied by A and L, respectively. Some humanized antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:58 and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:59. In some humanized antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:57 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:59. In some humanized antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:57 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:60. In some humanized antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:58 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:59. In some humanized antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:58 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:60.

Any of the above described antibodies can be an intact antibody, a single-chain antibody, Fab, or Fab′2 fragment. In any of the antibodies, the mature light chain variable region can be fused to a light chain constant region and the mature heavy chain variable region can be fused to a heavy chain constant region. Optionally, the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fcγ receptor relative to the natural human heavy chain constant region. Optionally the heavy chain constant region is of IgG1 isotype. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO:61 and/or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO:62. Optionally, the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO:61, 89, or 101 and/or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO:62 or 90. Any of the above antibodies can be conjugated to a therapeutic or cytotoxic agent, such as saporin or a radioisotope.

The invention further provides pharmaceutical composition comprising any of the above antibodies and a pharmaceutically acceptable carrier.

The invention further provides a nucleic acid encoding the heavy and/or light chain(s) of any of the above described antibodies, such as any of SEQ ID NOS:63-64, 67-68, 71-73, and 77-80. The invention further provides a nucleic acid encoding the heavy and/or light chain(s) of any of the above described antibodies, such as any of SEQ ID NOS:63-64, 67-68, 71-73, 77-80, 92-93, 99-100, or 102.

The invention further provides a recombinant expression vector comprising a nucleic acid as above described and a host cell transformed with such a recombinant expression vector.

The invention further provides a method of humanizing an antibody, the method comprising: (a) determining the sequences of the heavy and light chain variable regions of a mouse antibody; (b) synthesizing a nucleic acid encoding a humanized heavy chain comprising CDRs of the mouse antibody heavy chain and a nucleic acid encoding a humanized light chain comprising CDRs of the mouse antibody light chain; (c) expressing the nucleic acids in a host cell to produce a humanized antibody; wherein the mouse antibody is 15F7, 6C12, or 13G10.

The invention further provides a method of producing a humanized, chimeric, or veneered antibody, the method comprising: (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the antibody, so that the cells secrete the antibody; and (b) purifying the antibody from cell culture media; wherein the antibody is an antibody is a humanized, chimeric, or veneered form of 15F7, 6C12, or 13G10.

The invention further provides a method of producing a cell line producing a humanized, chimeric, or veneered antibody, the method comprising: (a) introducing a vector encoding heavy and light chains of an antibody and a selectable marker into cells; (b) propagating the cells under conditions to select for cells having increased copy number of the vector; (c) isolating single cells from the selected cells; and (d) banking cells cloned from a single cell selected based on yield of antibody; wherein the antibody is a humanized, chimeric, or veneered form of 15F7, 6C12, or 13G10. Optionally, the method further comprises propagating the cells under selective conditions and screening for cell lines naturally expressing and secreting at least 100 mg/L/10⁶ cells/24 h.

The invention further provides a method of treating or effecting prophylaxis of a cancer in a patient having or at risk for the cancer, the method comprising administering to the patient an effective regime of any of the above described antibodies. In some methods, the patient has a cancer, and the cancer is melanoma, breast cancer, lung cancer, or colorectal cancer.

The invention further provides a method of inhibiting cell adhesion in a biological sample, the method comprising contacting the biological sample with an effective amount of any of the above described antibodies. In some methods, the cell adhesion is mediated by the LG4-5 modules of the G domain of laminin α4. In some methods, the biological sample comprises cancer cells.

The invention further provides a method for detecting the presence of a cancer, such as melanoma, in a biological sample, the method comprising: (a) contacting the biological sample with any of the above antibodies; (b) detecting binding of the antibody to the biological sample; (c) contacting a control sample with the antibody; (d) detecting binding of the antibody to the control sample; and (e) comparing binding of the antibody to the biological sample with binding of the antibody to the control sample, whereby increased binding of the antibody to the biological sample compared to the control sample indicates the presence of cancer in the biological sample. Optionally the control sample and the biological sample comprise cells of the same tissue origin. Optionally binding of the antibody to the biological sample is at least 2-fold or 5-fold greater than the binding of the antibody to the control sample.

The invention further provides a method of evaluating the efficacy of a therapeutic agent in a patient diagnosed with a cancer, the method comprising: (a) contacting a first biological sample from the patient, obtained prior to treatment with the therapeutic agent, with any of the above antibodies; (b) detecting binding of the antibody to the first biological sample; (c) contacting a second biological sample from the patient, obtained following treatment with the therapeutic agent, with the antibody; (d) detecting binding of the antibody to the second biological sample; (e) comparing binding of the antibody to the first biological sample with binding of the antibody to the second biological sample, whereby decreased binding of the antibody to the second biological sample compared to the first biological sample indicates that the therapeutic agent is effective in treating the cancer in the patient.

The invention further provides a method of inhibiting binding of laminin α4 to a heparan sulfate proteoglycan in a biological sample, the method comprising contacting the biological sample with any of the above antibodies. Optionally, the heparan sulfate proteoglycan is syndecan-1, -2, -3, or -4 or glypican, a betaglycan, CD44, perlecan, agrin, or collagen XVIII.

The invention further provides a method of treating or effecting prophylaxis of a disease in which the LG4-5 modules of the G domain of laminin α4 contribute to progression of the disease, the method comprising administering to a patient having or at risk of the disease an effective regime of any of the above antibodies. Optionally, the disease is a cancer, such as melanoma, breast cancer, lung cancer, or colorectal cancer. Optionally the LG4-5 modules of the G domain of laminin α4 contribute to progression of the disease by means of interaction with a heparan sulfate proteoglycan. Optionally, the heparan sulfate proteoglycan is syndecan-1, -2, -3, or -4. Optionally, the disease is psoriasis, sarcoidosis, multiple sclerosis, or psoriatric arthritis.

The invention further provides a method of treating or effecting prophylaxis of an autoimmune disease in a patient having or at risk for the autoimmune disease, the method comprising administering to the patient an effective regime of any of the above described antibodies. Optionally, the disease is diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, celiac disease, psoriasis, psoriatic arthritis, sarcoidosis, ankylosing spondylitis, Sjogren's syndrome, or uveitis.

The invention further provides a method of inhibiting angiogenesis in a patient, the method comprising administering to a patient an effective regime of any of the above described antibodies. Optionally the patient has a cancer.

The invention further provides a method of treating or effecting prophylaxis of obesity or an obesity-related disease in a patient having or at risk for obesity or the obesity-related disease, the method comprising administering to the patient an effective regime of any of the above described antibodies. Optionally, the obesity-related disease is non-alcoholic steatohepatitis (NASH), Prader-Willi syndrome, craniopharyngioma, Bardet-Biedl syndrome, Cohen syndrome, or MOMO syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding as assessed by flow cytometry of a LG1-3-specific LAMA4 antibody and the 6C12, 13G10, and 15F7 antibodies to 293 cells displaying LAMA4 fragments containing LG1-5, LG1-3, and LG4-5.

FIG. 2A-C show relative binding and on/off rates for the 15F7, 6C12, and 13G10 antibodies, respectively, as assessed by ForteBio.

FIG. 3 shows the capacity of serially diluted 6C12, 13G10 and 15F7 antibodies to bind to LAMA4-displaying 293 cells as assessed by flow cytometry.

FIG. 4A-C show assessment of binding by FACS analysis of the 6C12, 13G10, and 15F7 antibodies, respectively, to LAMA4-displaying 293 cells in the presence of decreasing ratios (5:1, 1:1, and 1:5) of competing blocking antibodies.

FIG. 5A-C show staining of tumor microarray slides for human breast, colon, and lung tumors, respectively, with the 15F7 antibody.

FIG. 6 shows a cell adhesion assay in which recombinant LG4-5-coated ELISA plates were incubated with 20 ug/ml 15F7, 6C12, 13G10, and mouse IgG1 control and then assayed for their ability to bind to WM-266-4 human melanoma cells.

FIG. 7 shows toxicity to WM-266-4 cells when incubated with the 15F7 antibody and a saporin-conjugated anti-mouse secondary antibody.

FIG. 8 shows the capacity of serially diluted chimeric 15F7 and humanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 to bind to LAMA4-displaying cells as assessed by flow cytometry.

FIG. 9 shows binding as assessed by flow cytometry of chimeric 15F7 and humanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 to 293 cells displaying LAMA4 fragments containing LG1-5, LG1-3, and LG4-5.

FIG. 10A-B show relative binding and on/off rates for m15F7, chimeric 15F7 and humanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 as assessed by ForteBio, with the anti-His sensor being loaded with His-LAMA4 followed by association and dissociation of the 15F7 antibodies in 10A, and the goat anti-human Fc sensor being loaded with the antibodies followed by association and dissociation of His-LAMA4 in 10B.

FIGS. 11A & B show ratios of the relative levels of pAkt to Akt in human melanoma cells treated with laminin 411 or BSA control and with 15F7 or mIgG1 control. FIG. 11A shows the ratio for each individual sample, and FIG. 11B shows averages and standard errors for each group (n=3).

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

SEQ ID NO:1 sets forth the amino acid sequence of laminin α4 as provided by UniProt Number Q16363.

SEQ ID NO:2 sets forth the amino acid sequence of laminin α4 as provided by GenBank Accession Number NP001098676.

SEQ ID NO:3 sets forth the amino acid sequence of laminin α4 as provided by GenBank Accession Number NP001098677.

SEQ ID NO:4 sets forth the amino acid sequence of the G domain of laminin α4.

SEQ ID NO:5 sets forth the amino acid sequence of the LG1 module of the G domain of laminin α4.

SEQ ID NO:6 sets forth the amino acid sequence of the LG2 module of the G domain of laminin α4.

SEQ ID NO:7 sets forth the amino acid sequence of the LG3 module of the G domain of laminin α4.

SEQ ID NO:8 sets forth the amino acid sequence of the LG1-3 modules of the G domain of laminin α4.

SEQ ID NO:9 sets forth the amino acid sequence of the LG4 module of the G domain of laminin α4.

SEQ ID NO:10 sets forth the amino acid sequence of the LG5 module of the G domain of laminin α4.

SEQ ID NO:11 sets forth the amino acid sequence of the LG4-5 modules of the G domain of laminin α4.

SEQ ID NO:12 sets forth the amino acid sequence of syndecan-1 as provided by UniProt Number P18827.

SEQ ID NO:13 sets forth the amino acid sequence of syndecan-2 as provided by UniProt Number P34741.

SEQ ID NO:14 sets forth the amino acid sequence of syndecan-3 as provided by UniProt Number 075056.

SEQ ID NO:15 sets forth the amino acid sequence of syndecan-4 as provided by UniProt Number P31431.

SEQ ID NO:16 sets forth the amino acid sequence of mouse 15F7 mature heavy chain variable region.

SEQ ID NO:17 sets forth the amino acid sequence of mouse 15F7 mature light chain variable region.

SEQ ID NO:18 sets forth the amino acid sequence of the 15F7 heavy chain variable region signal peptide.

SEQ ID NO:19 sets forth the amino acid sequence of the 15F7 light chain variable region signal peptide.

SEQ ID NO:20 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 15F7 heavy chain.

SEQ ID NO:21 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 15F7 heavy chain.

SEQ ID NO:22 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 15F7 heavy chain.

SEQ ID NO:23 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 15F7 light chain.

SEQ ID NO:24 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 15F7 light chain.

SEQ ID NO:25 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 15F7 light chain.

SEQ ID NO:26 sets forth the amino acid sequence of mouse 6C12 mature heavy chain variable region.

SEQ ID NO:27 sets forth the amino acid sequence of mouse 6C12 mature light chain variable region, version 1.

SEQ ID NO:28 sets forth the amino acid sequence of the 6C12 heavy chain variable region signal peptide.

SEQ ID NO:29 sets forth the amino acid sequence of the 6C12 light chain variable region signal peptide.

SEQ ID NO:30 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 6C12 heavy chain.

SEQ ID NO:31 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 6C12 heavy chain.

SEQ ID NO:32 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 6C12 heavy chain.

SEQ ID NO:33 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 6C12 light chain, version 1.

SEQ ID NO:34 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 6C12 light chain.

SEQ ID NO:35 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 6C12 light chain.

SEQ ID NO:36 sets forth the amino acid sequence of mouse 13G10 mature heavy chain variable region, version 1.

SEQ ID NO:37 sets forth the amino acid sequence of mouse 13G10 mature heavy chain variable region, version 2.

SEQ ID NO:38 sets forth the amino acid sequence of mouse 13G10 mature light chain variable region.

SEQ ID NO:39 sets forth the amino acid sequence of the 13G10 heavy chain variable region signal peptide, version 1.

SEQ ID NO:40 sets forth the amino acid sequence of the 13G10 heavy chain variable region signal peptide, version 2.

SEQ ID NO:41 sets forth the amino acid sequence of the 13G10 light chain variable region signal peptide.

SEQ ID NO:42 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:43 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:44 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:45 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:46 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:47 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:48 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 13G10 light chain.

SEQ ID NO:49 sets forth the amino acid sequence of CDR2, as defined by Kabat, of the mouse 13G10 light chain.

SEQ ID NO:50 sets forth the amino acid sequence of CDR3, as defined by Kabat, of the mouse 13G10 light chain.

SEQ ID NO:51 sets forth the amino acid sequence of a human VH acceptor FR as provided by NCBI Accession Code ACF36857.1, version 1.

SEQ ID NO:52 sets forth the amino acid sequence of a human VH acceptor FR as provided by NCBI Accession Code BAC01530.1, version 1.

SEQ ID NO:53 sets forth the amino acid sequence of a human VL acceptor FR as provided by NCBI Accession Code AAY33350.1.

SEQ ID NO:54 sets forth the amino acid sequence of a human VL acceptor FR as provided by NCBI Accession Code BAC01583.1, version 1.

SEQ ID NO:55 sets forth the amino acid sequence of humanized 15F7 heavy chain variable region with no backmutations or other mutations.

SEQ ID NO:56 sets forth the amino acid sequence of humanized 15F7 light chain variable region with no backmutations or other mutations.

SEQ ID NO:57 sets forth the amino acid sequence of humanized 15F7 heavy chain variable region version 1 (H1).

SEQ ID NO:58 sets forth the amino acid sequence of humanized 15F7 heavy chain variable region version 2 (H2).

SEQ ID NO:59 sets forth the amino acid sequence of humanized 15F7 light chain variable region version 1 (L1).

SEQ ID NO:60 sets forth the amino acid sequence of humanized 15F7 light chain variable region version 2 (L2).

SEQ ID NO:61 sets forth the amino acid sequence of an exemplary human IgG1 constant region.

SEQ ID NO:62 sets forth the amino acid sequence of an exemplary human kappa light chain constant region without a N-terminal arginine.

SEQ ID NO:63 sets forth the nucleic acid sequence of the murine 15F7 heavy chain variable region.

SEQ ID NO:64 sets forth the nucleic acid sequence of the murine 15F7 light chain variable region version.

SEQ ID NO:65 sets forth the nucleic acid sequence of the 15F7 heavy chain variable region signal peptide.

SEQ ID NO:66 sets forth the nucleic acid sequence of the 15F7 light chain variable region signal peptide.

SEQ ID NO:67 sets forth the nucleic acid sequence of the murine 6C12 heavy chain variable region.

SEQ ID NO:68 sets forth the nucleic acid sequence of the murine 6C12 light chain variable region, version 1.

SEQ ID NO:69 sets forth the nucleic acid sequence of the 6C12 heavy chain variable region signal peptide.

SEQ ID NO:70 sets forth the nucleic acid sequence of the 6C12 light chain variable region signal peptide.

SEQ ID NO:71 sets forth the nucleic acid sequence of the murine 13G10 heavy chain variable region, version 1.

SEQ ID NO:72 sets forth the nucleic acid sequence of the murine 13G10 heavy chain variable region, version 2.

SEQ ID NO:73 sets forth the nucleic acid sequence of the murine 13G10 light chain variable region.

SEQ ID NO:74 sets forth the nucleic acid sequence of the 13G10 heavy chain variable region signal peptide, version 1.

SEQ ID NO:75 sets forth the nucleic acid sequence of the 13G10 heavy chain variable region signal peptide, version 2.

SEQ ID NO:76 sets forth the nucleic acid sequence of the 13G10 light chain variable region signal peptide.

SEQ ID NO:77 sets forth the nucleic acid sequence of humanized 15F7 heavy chain variable region version 1 (H1).

SEQ ID NO:78 sets forth the nucleic acid sequence of humanized 15F7 heavy chain variable region version 2 (H2).

SEQ ID NO:79 sets forth the nucleic acid sequence of humanized 15F7 light chain variable region version 1 (L1), version 1.

SEQ ID NO:80 sets forth the nucleic acid sequence of humanized 15F7 light chain variable region version 2 (L2).

SEQ ID NO:81 sets forth the nucleic acid sequence of the G domain of laminin α4.

SEQ ID NO:82 sets forth the nucleic acid sequence of the LG1 module of the G domain of laminin α4.

SEQ ID NO:83 sets forth the nucleic acid sequence of the LG2 module of the G domain of laminin α4.

SEQ ID NO:84 sets forth the nucleic acid sequence of the LG3 module of the G domain of laminin α4.

SEQ ID NO:85 sets forth the nucleic acid sequence of the LG1-3 modules of the G domain of laminin α4.

SEQ ID NO:86 sets forth the nucleic acid sequence of the LG4 module of the G domain of laminin α4.

SEQ ID NO:87 sets forth the nucleic acid sequence of the LG5 module of the G domain of laminin α4.

SEQ ID NO:88 sets forth the nucleic acid sequence of the LG4-5 modules of the G domain of laminin α4.

SEQ ID NO:89 sets forth the amino acid sequence of an exemplary human IgG1 constant region of the IgG1 G1m3 allotype.

SEQ ID NO:90 sets forth the amino acid sequence of an exemplary human kappa light chain constant region with a N-terminal arginine.

SEQ ID NO:91 sets forth the amino acid sequence of an exemplary human IgG1 constant region without a C-terminal lysine.

SEQ ID NO:92 sets forth the nucleic acid sequence of an exemplary human IgG1 constant region of the IgG1 G1m3 allotype.

SEQ ID NO:93 sets forth the nucleic acid sequence of an exemplary human kappa light chain constant region with a N-terminal arginine.

SEQ ID NO:94 sets forth the amino acid sequence of mouse 6C12 mature light chain variable region, version 2.

SEQ ID NO:95 sets forth the amino acid sequence of CDR1, as defined by Kabat, of the mouse 6C12 light chain, version 2.

SEQ ID NO:96 sets forth the amino acid sequence of a human VH acceptor FR as provided by NCBI Accession Code ACF36857.1, version 2.

SEQ ID NO:97 sets forth the amino acid sequence of a human VH acceptor FR as provided by NCBI Accession Code BAC01530.1, version 2.

SEQ ID NO:98 sets forth the amino acid sequence of a human VL acceptor FR as provided by NCBI Accession Code BAC01583.1, version 2.

SEQ ID NO:99 sets forth the nucleic acid sequence of the murine 6C12 light chain variable region, version 2.

SEQ ID NO:100 sets forth the nucleic acid sequence of humanized 15F7 light chain variable region version 1 (L1), version 2.

SEQ ID NO:101 sets forth the amino acid sequence of an exemplary human IgG1 constant region of the IgG1 G1m3 allotype.

SEQ ID NO:102 sets forth the nucleic acid sequence of an exemplary human kappa light chain constant region without a N-terminal arginine.

DEFINITIONS

Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that an antibody or other biologically entity is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/w pure of interfering proteins and contaminants from production or purification. Often an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.

Specific binding of an antibody to its target antigen means an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.

The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.

The term “antibody” includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes a bispecific antibody and/or a humanized antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy/light chain pairs include a humanized 15F7 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on laminin α4 than that bound by 15F7.

In some bispecific antibodies, one heavy chain light chain pair is a humanized 15F7 antibody as further disclosed below and the heavy light chain pair is from an antibody that binds to a receptor expressed on the blood brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Friden et al., PNAS 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such a bispecific antibody can be transferred cross the blood brain barrier by receptor-mediated transcytosis. Brain uptake of the bispecific antibody can be further enhanced by engineering the bi-specific antibody to reduce its affinity to the blood brain barrier receptor. Reduced affinity for the receptor resulted in a broader distributioin in the brain (see, e.g., Atwal. et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (5) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region. Examples of platforms useful for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the references antibody by at least 75%, 90% or 99%. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.

The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has at least one known risk-factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.

The term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny. Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

The term “control sample” refers to a biological sample not known or suspected to include cancerous cells, or at least not known or suspect to include cancerous cells of a given type. Control samples can be obtained from individuals not afflicted with cancer or a specifically chosen type of cancer. Alternatively, control samples can be obtained from patients afflicted with cancer or a specifically chosen type of cancer. Such samples can be obtained at the same time as a biological sample thought to comprise the cancer or on a different occasion. A biological sample and a control sample can both be obtained from the same tissue (e.g., a tissue section containing both tumor tissue and surrounding normal tissue). Preferably, control samples consist essentially or entirely of normal, healthy cells and can be used in comparison to a biological sample thought to comprise cancer cells or a particular type of cancer cells. Preferably, the cells in the control sample have the same tissue origin as the cancer cells thought to be in the biological sample (e.g., lung or brain). Preferably, the cancer cells thought to be in the biological sample arise from the same cell type (e.g., neuronal, epithelial, mesenchymal, hematopoietic) as the type of cells in the control sample.

The term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such as altered gait, as perceived by the subject. A “sign” refers to objective evidence of a disease as observed by a physician.

For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.

Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” an antibody may contain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value.

Statistical significance means p≦0.05.

The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” can include a plurality of compounds, including mixtures thereof.

DETAILED DESCRIPTION I. General

The invention provides antibodies that specifically bind to the LG4-5 modules of the G domain of laminin α4. The antibodies have the ability selectively to stain cancer cells relative to normal tissue. The antibodies also have the ability to inhibit laminin-α4-mediated cancer or tumor cell adhesion. The antibodies can be used for detecting cancer, evaluating the efficacy of a cancer therapy, treating cancer, and treating obesity or obesity-related diseases, among other applications.

II. Target Molecules

Laminins are a family of extracellular matrix glycoproteins and are the major non-collagenous constitutent of basement membranes. They have been reported to be involved in biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth, and metastasis, among other processes. Laminins are heterotrimeric proteins of three chains: an alpha chain, a beta chain, and a gamma chain. The three chains form a cruciform structure consisting of three short arms, each formed by a different chain, and a long arm composed of all three chains. In mammals, five different alpha chains, three different beta chains, and three different gamma chains have been identified that can assemble into fifteen different heterotrimeric combinations.

The laminin alpha chains have a large C-terminal globular domain (G domain) that has five tandem homologous laminin G-like modules (LG1-5) of about 200 amino acids. For example, the G domain of laminin α4 is defined by UniProt sequence Q16363 as amino acid positions 833-1820 (SEQ ID NO:4), and the five LG modules of laminin α4 are defined by UniProt sequence Q16363 as follows: LG1 (SEQ ID NO:5) includes amino acid positions 833-1035, LG2 (SEQ ID NO:6) includes amino acid positions 1047-1227, LG3 (SEQ ID NO:7) includes amino acid positions 1234-1402, LG4 (SEQ ID NO:9) includes amino acid positions 1469-1640, and LG5 (SEQ ID NO:10) includes amino acid positions 1647-1820. In some cases, the G domain can be SEQ ID NO:4; in other cases it can include amino acid positions 833-1820 of UniProt sequence Q16363. In some cases, the LG1 module can be SEQ ID NO:5; in other cases it can include amino acid positions 833-1035 of UniProt sequence Q16363. In some cases, the LG2 module can be SEQ ID NO:6; in other cases it can include amino acid positions 1047-1227 of UniProt sequence Q16363. In some cases, the LG3 module can be SEQ ID NO:7; in other cases it can include amino acid positions 1234-1402 of UniProt sequence Q16363. In some cases, the LG4 module can be SEQ ID NO:9; in other cases it can include amino acid positions 1469-1640 of UniProt sequence Q16363. In some cases, the LG5 module can be SEQ ID NO:10; in other cases it can include amino acid positions 1647-1820 of UniProt sequence Q16363. The LG1-3 modules (SEQ ID NO:8) are connected to the LG4-5 modules (SEQ ID NO:11) by a linker domain. The laminin α4 chain (also known as LAMA4, laminin subunit α4, laminin-14 subunit alpha, laminin-8 subunit alpha, and laminin-9 subunit alpha) is 200 kDa and is the shortest variant. Compared to the α1, α2, and α5 chains, laminin α4 has a truncated N-terminus. Laminin α4 is widely distributed both in adults and during development. It is present in laminin-8 (laminin 411 or alpha4/beta1/gamma1), laminin-9 (laminin 421 or alpha4/beta2/gamma1), and laminin-14 (laminin 411 or alpha4/beta1/gamma1).

Unless otherwise apparent from context, reference to laminin α4 or its fragments, domains, or modules includes the natural human amino acid sequences including isoforms and allelic variants thereof. Exemplary human sequences are designated UniProt Number Q16363 and GenBank Accession Numbers NP001098676 and NP001098677 (SEQ ID NOS:1, 2, and 3, respectively). Some antibodies bind to an epitope within the LG4-5 modules of the G domain of laminin α4. The epitope can be in LG4, in LG5, or split so that residues forming the epitope come from both LG4 and LG5.

Laminin α4 has been reported to be a binding partner for syndecans. For example, cells overexpressing syndecan-2 or -4 have been reported to bind to the LG4 module of the G domain of laminin α4. See Matsuura et al., J. Invest. Dermatol. 122:614-620 (2004). Syndecans are transmembrane heparan sulfate proteoglycan receptors that constitute the major physiological form of heparan sulfate on the cell surface. There are four reported members of the syndecan protein family: syndecan-1 (also known as SYND1, SDC1, SDC, or CD138), syndecan-2 (also known as SYND2, SDC2, fibroglycan, heparan sulfate proteoglycan core protein, HSPG, HSPG1, and CD362), syndecan-3 (also known as SYND3 and SDC3), and syndecan-4 (also known as SYND4, SDC4, amphiglycan, and ryudocan core protein). Syndecans have a short cytoplasmic domain, a single-span transmembrane domain, and an extracellular domain. They have been reported to bind to growth factors, cytokines, and extracellular matrix proteins. Syndecans have many reported biological roles including regulating cell growth, differentiation, angiogenesis, and adhesion, among other processes. Unless otherwise apparent from context, reference to a syndecan or its fragments or domains includes the natural human amino acid sequences including isoforms and allelic variants thereof. Exemplary human sequences are designated UniProt Numbers P18827 (syndecan-1), P34741 (syndecan-2), 075056 (syndecan-3), and P31431 (syndecan-4) (SEQ ID NOS:12-15, respectively).

The LG4 and LG5 modules of the G domain of laminin α4 also bind to other heparan sulfate proteoglycans including cell surface proteins such as glypicans, betaglycans, and CD44 (e.g., Swiss Prot. P16070), and matrix proteins such as perlecan (e.g., Swiss Prot. P98160), agrin (e.g., Swiss Prot. Q00468), and collagen XVIII (e.g., Swiss Prot. P39060). Binding of the LG4-5 modules of the G domain of laminin α4 to any or all of these or other proteoglycans may contribute to its growth promoting characteristics, inflammation, angiogenesis, and growth and metastasis of cancers.

III. Cancers

The above target molecules are involved in the development and/or progression of cancers. Cancer is a physiological condition in mammals that is typically characterized by unregulated cell growth and proliferation. Cancers can be hematopoietic malignancies or solid tumors, i.e., masses of cells that result from excessive cell growth or proliferation, including pre-cancerous legions. Metastatic cancer refers to a cancer that has spread from the place where it first started to another place in the body. Tumors formed by metastatic cancer cells are called a metastatic tumor or a metastasis, which is a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancer has the same name and same type of cancer cells as the original, or primary, cancer. Examples solid tumors include melanoma, carcinoma, blastoma, and sarcoma. Hematologic malignancies include leukemia or lymphoid malignancies, such as lymphoma. More particular examples of such cancers include squamous cell cancer or carcinoma, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

IV. Antibodies

A. Binding Specificity and Functional Properties

The invention provides antibodies binding to epitopes within the LG4-5 modules of the G domain of laminin α4. For example, as defined by laminin α4 UniProt sequence Q16363, LG4 (SEQ ID NO:9) includes amino acid positions 1469-1640, LG5 (SEQ ID NO:10) includes amino acid positions 1647-1820, and LG4-5 (SEQ ID NO:11) includes amino acid positions 1469-1820. The epitope can be in LG4, in LG5, or split so that residues forming the epitope come from both LG4 and LG5. The epitope can be in particular segments within LG4-5, such as segments from laminin α4 UniProt sequence Q16363 ranging from positions 1469-1519, 1520-1570, 1571-1621, 1622-1672, 1673-1723, 1724-1774, and 1775-1820. The epitope can be linear, such as an epitope of, for example, 2-5, 3-5, 3-10, 3-15, 3-20, 5-10, 5-15, or 5-20 contiguous amino acids from LG4, LG5, LG4-5, or any of the segments or pairs of adjoining segments specified above. The epitope can also be a conformational epitope including, for example, 2-5, 3-5, 3-10, 3-15, 3-20, 5-10, 5-15, or 5-20 non-contiguous amino acids from any combination of LG4, LG5, LG4-5, and any of the segments specified above.

Antibodies designated 15F7, 6C12, and 13G10 are three such exemplary mouse antibodies. These three monoclonal antibodies each specifically bind to an epitope within the LG4 and/or LG5 modules of the G domain of laminin α4. These antibodies are further characterized by their lack of significant binding to the LG1-3 modules of the G domain of laminin α4 (e.g., same within experimental error as an irrelevant control antibody, or binding that is at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold less (e.g., as measured by a flow cytometric binding assay) than an antibody specific for the LG1-3 modules). Some antibodies are characterized by their lack of significant binding to other laminin alpha chains, e.g., laminin α1, laminin α2, laminin α3, and laminin α5 (e.g., same within experimental error as an irrelevant control antibody, or binding that is at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold less (e.g., as measured by a flow cytometric binding assay) than an antibody specific for the relevant other laminin alpha chain). Some antibodies are further characterized by their capacity selectively to bind a biological sample comprising cancer cells (e.g., a sample of tumor tissue) when compared to control sample (e.g., binding of the antibody to the biological sample is at least 1.5-fold, 2-fold, 5-fold, or 10-fold greater than binding of the antibody to the control sample), as shown in Example 2 with melanoma samples. Preferably, the control sample and the biological sample comprise cells of the same tissue origin. Ability to bind to specific proteins, modules, or domains can be demonstrated using exemplary assay formats provided in the examples. Likewise, ability to selectively bind and stain tumor tissue can be demonstrated using exemplary assay formats provided in the examples.

Some antibodies binding within LG4-5 lack capacity to inhibit binding of laminin α4 to MCAM (i.e., extent of inhibition the same within experimental error as an irrelevant control antibody). Some antibodies inhibit binding of laminin α4 to a syndecan, such as syndecan-1, syndecan-2, syndecan-3, or syndecan-4. Some antibodies inhibit binding of laminin α4 to other heparan sulfate proteoglycans including several other cell surface proteins including any or all of glypicans, betaglycans, and CD44, and matrix proteins such as perlecan, agrin, and collagen XVIII. Some of the disclosed antibodies can inhibit binding of the LG4-5 modules of the G domain of laminin α4 to any or all of these proteoglycans, and consequently or otherwise inhibit angiogenesis, inflammation and growth and metastasis of cancers.

Inhibition of binding of laminin α4 to a binding partner, for example a syndecan or other proteoglycan, can be tested in a binding assay in which an antibody is pre-incubated with recombinant laminin α4 protein, laminin-α4-positive tissue, or laminin-α4-displaying cells, after which recombinant proteoglycan or proteoglycan-expressing cells are then assessed for their ability to bind to laminin α4. Optionally, inhibition of a test antibody can be demonstrated in comparison to an irrelevant control antibody not binding within the LG4-5 modules of the G domain of laminin α4 or in comparison to vehicle lacking any antibody.

Some antibodies have the capacity to inhibit laminin-α4-mediated cell adhesion of cancer cells, preferably cell adhesion mediated by the LG4-5 modules of the G domain of laminin α4. An exemplary cell adhesion assay is described in the examples.

Some antibodies also have the capacity to inhibit laminin-α4-induced pAkt activation. An exemplary assay is described in the examples.

Inhibition means an inhibition of at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75% or 30%-70%) of binding, cell adhesion and/or other functional activity mediated by laminin α4, either alone or in combination with a syndecan or anything else contributing (e.g., other proteoglycan) to any of its functional activities. Inhibition can usually demonstrated when the antibody is present at a concentration of about 20 ug/ml. The antibodies can show inhibition of at least 30% of laminin-α4-mediated cell adhesion, such as, for example, cell adhesion mediated by the LG4-5 modules of the G domain of laminin α4.

Preferred antibodies inhibit cancer development or progression, or any aspect thereof, such as metastasis, as shown in an animal model or clinical trial. Animal models of cancer in which human cancer cells are injected into an immunodeficient laboratory animal, such as a mouse or rat, or transgenic models in which a laboratory animal expresses a human oncogene or has a knocked out tumor suppressor gene, are widely available. In addition, cell-based assays for particular characteristics of cancer cells, such as proliferation assays, growth assays, survival assays, migration assays, invasion assays, and others, are widely available.

Some antibodies bind to the same or overlapping epitope as an antibody designated 15F7, 6C12, or 13G10. The sequences of the heavy and light chain mature variable regions of these antibodies are designated SEQ ID NOS:16 and 17, 26 and 27, and 36/37 and 38, respectively. Another version of the light chain mature variable region of 6C12 is SEQ ID NO:94. Other antibodies having such a binding specificity can be produced by immunizing mice with laminin α4, or a portion thereof including the desired epitope, and screening resulting antibodies for binding to the LG4-5 modules of the G domain of laminin α4, optionally in competition with 15F7, 6C12, or 13G10. Antibodies identified by such assays can then be screened for ability to specifically bind to the LG4-5 modules but not the LG1-3 modules of the G domain of laminin α4 as described in the examples or otherwise. Antibodies can also be screened for ability to selectively stain tumor tissue as described in the examples or otherwise. Antibodies can also be screened for ability to inhibit laminin-α4-mediated tumor cell adhesion as described in the examples or otherwise.

Antibodies binding to an epitope that includes one or more specified residues can be generated by immunizing with a fragment of laminin α4 that includes these one or more residues. The fragment can, for example, have no more than 100, 50, 25, 10 or 5 contiguous amino acids from SEQ ID NO:11. Such fragments usually have at least 5, 6, 7, 8 or 9 contiguous residues of SEQ ID NO:11. The fragments can be linked to a carrier that helps elicit an antibody response to the fragment and/or be combined with an adjuvant that helps elicit such a response. Alternatively, antibodies binding to a desired residue can be obtained by immunizing with a full-length laminin α4 (SEQ ID NO:1) or the full-length G domain of laminin α4 (SEQ ID NO:4) or the LG4-5 modules of the G domain of laminin α4 (SEQ ID NO:11) or fragments of any of these. Such antibodies can then be screened for differential binding to versions of laminin α4 containing different LG modules of the G domain, such as LG1-3, LG1-5, LG4-5, LG4, or LG5 (SEQ ID NOS:8, 4, 11, 9, and 10, respectively) or differential binding to wild type laminin α4 compared with mutants of specified residues. The screen against versions of laminin α4 with different LG modules of the G domain maps antibody binding to certain LG modules within the G domain of laminin α4. The screen against mutants more precisely defines the binding specificity to allow identification of antibodies whose binding is inhibited by mutagenesis of particular residues and which are likely to share inhibitor properties of other exemplified antibodies.

Human antibodies having the binding specificity of a selected murine antibody (e.g., 15F7, 6C12, or 13G10) can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions can for example be obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding for the LG4-5 modules of the G domain of laminin α4 (e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) is selected. The heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained for example from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding for the LG4-5 modules of the G domain of laminin α4 are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 15F7, 6C12, or 13G10. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 15F7, 6C12, or 13G10 in amino acid sequence of the mature heavy and/or light chain variable regions and maintain its functional properties, and/or which differ from the respective antibody by a small number of functionally inconsequential amino acid substitutions (e.g., conservative substitutions), deletions, or insertions are also included in the invention. Monoclonal antibodies having at least one or all six CDR(s) as defined by Kabat that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 15F7, 6C12, or 13G10 are also included.

The invention also provides antibodies having some or all (e.g., 3, 4, 5, and 6) CDRs entirely or substantially from 15F7, 6C12, or 13G10. Such antibodies can include a heavy chain variable region that has at least two, and usually all three, CDRs entirely or substantially from the heavy chain variable region of 15F7, 6C12, or 13G10 and/or a light chain variable region having at least two, and usually all three, CDRs entirely or substantially from the light chain variable region of 15F7, 6C12, or 13G10. The antibodies can include both heavy and light chains. A CDR is substantially from a corresponding 15F7, 6C12, or 13G10 CDR when it contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDRH2 (when defined by Kabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 15F7, 6C12, or 13G10 in the amino acid sequence of the mature heavy and/or light chain variable regions and maintain their functional properties, and/or differ from 15F7, 6C12, or 13G10 by a small number of functionally inconsequential amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig, primate, rabbit or rat, against the LG4-5 modules of the G domain of laminin α4 can be accomplished by, for example, immunizing the animal with laminin α4 or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvant can be used as described below. Complete Freund's adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies. Antibodies are screened for specific binding to the LG4-5 modules of the G domain of laminin α4. Such screening can be accomplished by determining binding of an antibody to a collection of laminin α4 variants, such as laminin α4 variants containing the LG1-3 modules of the G domain, the LG1-5 modules of the G domain, and the LG4-5 modules of the G domain, and determining which laminin α4 variants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which the CDRs from a non-human “donor” antibody are grafted into human “acceptor” antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No. 6,407,213, Adair, U.S. Pat. No. 5,859,205 6,881,557, Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al., Mol. Immunol. 41: 863, 2004. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between a human acceptor sequence variable region frameworks and corresponding variable region frameworks of a donor antibody chain.

Examples of acceptor sequences for the heavy chain are the human mature heavy chain variable regions with NCBI accession codes ACF36857.1 and BAC01530.1 (SEQ ID NOS:51 and 52, respectively). Other versions of ACF36857.1 and BAC01530.1 are SEQ ID NOS:96 and 97, respectively. These acceptor sequences include two CDRs having the same canonical form as mouse 15F7 heavy chain. Examples of acceptor sequences for the light chain are the human mature light chain variable regions with NCBI accession codes AAY33350.1 and BAC01583.1 (SEQ ID NOS:53 and 54, respectively). Another version of BAC01583.1 is SEQ ID NO:98. These acceptor sequences include three CDRs having the same canonical form as mouse 15F7 light chain.

Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,     -   (2) is adjacent to a CDR region or within a CDR as defined by         Chothia but not Kabat,     -   (3) otherwise interacts with a CDR region (e.g. is within about         6 Å of a CDR region), (e.g., identified by modeling the light or         heavy chain on the solved structure of a homologous known         immunoglobulin chain), or     -   (4) is a residue participating in the VL-VH interface.

Framework residues from classes (1) through (3) as defined by Queen, U.S. Pat. No. 5,530,101, are sometimes alternately referred to as canonical and vernier residues. Framework residues that help define the conformation of a CDR loop are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues that support antigen-binding loop conformations and play a role in fine-tuning the fit of an antibody to antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Viol 224:487-499 (1992)).

Other framework residues that are candidates for substitution are residues creating a potential glycosylation site. Still other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins.

Exemplary humanized antibodies are humanized forms of the mouse 15F7 antibody, designated Hu15F7. The mouse antibody comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:16 and SEQ ID NO:17, respectively. The invention provides two exemplified humanized mature heavy chain variable regions: Hu15F7VHv1 (H1; SEQ ID NO:57) and Hu15F7VHv2 (H2; SEQ ID NO:58). The invention further provides two exemplified human mature light chain variable regions: Hu15F7VLv1 (L1; SEQ ID NO:59) and Hu15F7VLv2 (L2; SEQ ID NO:60).

For reasons such as possible influence on CDR conformation and/or binding to antigen, mediating interaction between heavy and light chains, interaction with the constant region, being a site for desired or undesired post-translational modification, being an unusual residue for its position in a human variable region sequence and therefore potentially immunogenic, getting aggregation potential, and other reasons, the following 20 variable region framework positions were considered as candidates for substitutions in the two exemplified human mature light chain variable regions and the two exemplified human mature heavy chain variable regions, as further specified in the examples: L8 (P8S), L42 (K42N), L45 (K45R), L49 (Y49S), L69 (T69K), L80 (P80T), H1 (Q1E), H20 (V20L), H27 (G27Y), H30 (530T), H38 (R38K), H40 (A40R), H41 (P41A), H48 (M48I), H66 (R66K), H67 (V67A), H69 (169L), H75 (T75S), H82A (S(82A)R), and H91 (Y91F). In addition, L104 (L104V) was considered as a candidate for substitution in the two exemplified human mature light chain variable regions.

Here, as elsewhere, the first-mentioned residue is the residue of a humanized antibody formed by grafting Kabat CDRs into a human acceptor framework, and the second-mentioned residue is a residue being considered for replacing such residue. Thus, within variable region frameworks, the first mentioned residue is human, and within CDRs, the first mentioned residue is mouse.

Exemplified antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions (e.g., VHv1/VLv1 or H1L1, VHv1/VLv2 or H1L2, VHv2/VLv1 or H2L1, and VHv2/VLv2 or H2L2). For example, the H1L1 antibody, which includes 14 heavy chain backmutations or other mutations and 6 light chain backmutations as described below, binds to laminin α4 and inhibits MCAM binding to laminin α4 at a level that is substantially the same as, if not superior to, a chimeric 19C12 antibody (see FIGS. 8-10). Comparable results are seen with the H1L2, H2L1, and H2L2 antibodies (see FIGS. 8-10).

The invention provides variants of the H1L1 humanized 15F7 antibody in which the humanized mature heavy chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to H1 (SEQ ID NO:57) and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to L1 (SEQ ID NO:59). In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 of the backmutations or other mutations in H1L1 are retained. The invention also provides variants of the H2L1 humanized 15F7 antibody in which the humanized mature heavy chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to H2 (SEQ ID NO:58) and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to L1 (SEQ ID NO:59). In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of the backmutations or other mutations in H2L1 are retained. In some antibodies, at least one of positions H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 in the Vh region is occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively. In some antibodies, positions H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 in the Vh region are occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively, such as in version H2. In some antibodies, at least one of positions H41 and H69 in the Vh region is occupied by A and L, respectively. In some antibodies, positions H41 and H69 in the Vh region are occupied by A and L, respectively, such as in version H1. In some antibodies, at least one of positions L42, L49, and L69 in the Vk region is occupied by N, S, and K, respectively. In some antibodies, positions L42, L49, and L69 in the Vk region are occupied by N, S, and K, respectively, such as in version L2. In some antibodies, at least one of positions L8, L45, and L80 in the Vk region is occupied by S, R, and T, respectively. In some antibodies, at least one of positions L8, L45, L80, and L104 in the Vk region is occupied by S, R, T, and V, respectively. In some antibodies, positions L8, L45, and L80 in the Vk region are occupied by S, R, and T, respectively, such as in version L1. In some antibodies, L104 is occupied by V, such as in version L2. The CDR regions of such humanized antibodies can be identical or substantially identical to the CDR regions of H1L1, which are the same as those of the mouse donor antibody. The CDR regions can be defined by any conventional definition (e.g., Chothia) but are preferably as defined by Kabat.

The invention also provides variants of the other exemplified Hu15F7 antibodies. Such variants have mature light and heavy chain variable regions showing at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the mature light and heavy chain variable regions of the exemplified humanized 15F7 H1L2, H2L1, or H2L2 antibodies. The CDR regions of such humanized antibodies can be identical or substantially identical to those of the mouse donor antibody. The CDR regions can be defined by any conventional definition (e.g., Chothia) but are preferably defined by Kabat. Variable regions framework positions are in accordance with Kabat numbering unless otherwise stated. Other such variants typically differ from the sequences of the exemplified Hu15F7 antibodies by a small number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) of replacements, deletions or insertions. Such differences are usually in the framework but can also occur in the CDRs.

A possibility for additional variation in humanized 15F7 variants is additional backmutations in the variable region frameworks. Many of the framework residues not in contact with the CDRs in the humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and even many potential CDR-contact residues are also amenable to substitution. Even amino acids within the CDRs may be altered, for example, with residues found at the corresponding position of the human acceptor sequence used to supply variable region frameworks. In addition, alternate human acceptor sequences can be used, for example, for the heavy and/or light chain. If different acceptor sequences are used, one or more of the backmutations recommended above may not be performed because the corresponding donor and acceptor residues are already the same without backmutations.

Preferably, replacements or backmutations in Hu15F7 (whether or not conservative) have no substantial effect on the binding affinity or potency of the humanized mAb, that is, its ability to bind to laminin α4 or the LG4-5 modules of the G domain of laminin α4 (e.g., the potency in some or all of the assays described in the present examples of the variant humanized 15F7 antibody is essentially the same, i.e., within experimental error, as that of murine 15F7 or H1L1).

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-human antibodies, particularly the 15F7, 6C12, or 13G10 antibodies of the examples.

A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions. Veneered forms of the 15F7 antibody are included in the invention.

E. Human Antibodies

Human antibodies against the LG4-5 modules of the G domain of laminin α4 are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of laminin α4, such as a laminin α4 variant containing only the LG4-5 modules of the G domain, as the target antigen, and/or by screening antibodies against a collection of laminin α4 variants, such as laminin α4 variants containing the LG1-3 modules of the G domain, the LG1-5 modules of the G domain, and the LG4-5 modules of the G domain.

Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., WO93/12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991)) and phage display methods (see, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and U.S. Pat. No. 5,565,332).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized (including veneered), or human antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent complement and/or cellular mediated cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 have complement-mediated cytotoxicity and human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. A human IgG1 constant region suitable for inclusion in the antibodies can have the sequence of SEQ ID NO:61. The C-terminal lysine of SEQ ID NO:61 can be omitted, in which case the IgG1 constant region has the amino acid sequence of SEQ ID NO:91. Light chain constant regions can be lambda or kappa. A human kappa light chain constant region suitable for inclusion in the antibodies can have the sequence of SEQ ID NO:90. SEQ ID NO:90 can be encoded by the nucleic acid sequence of SEQ ID NO:93. The N-terminal arginine of SEQ ID NO:90 can be omitted, in which case the kappa light chain constant region has the amino acid sequence of SEQ ID NO:62. SEQ ID NO:62 can be encoded by the nucleic acid sequence of SEQ ID NO:102. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes. Thus, for example, another heavy chain constant region is of the IgG1 G1m3 allotype and has the amino acid sequence of SEQ ID NO:89. SEQ ID NO:89 can be encoded by the nucleic acid sequence of SEQ ID NO:92. Another heavy chain constant region of the IgG1 G1m3 allotype has the amino acid sequence of SEQ ID NO:101. Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying polymorphic positions in natural allotypes.

One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position 250 and/or a Leu at position 428 (EU numbering) for increasing the half-life of an antibody. Substitution at any of positions 234, 235, 236 and/or 237 reduces affinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S. Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and 237 of human IgG1 can be used for reducing effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine. See, e.g., U.S. Pat. No. 5,624,821. In some antibodies, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. In some antibodies, a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgG1 is used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine, such as in SEQ ID NO:101. In some antibodies, the isotype is human IgG2 or IgG4.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g., hybridoma). For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide encompassing the translation initiation codon as the 5′ primer and a g2b constant regions specific 3′ primer. Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter “Schenk”). The sequences from multiple, independently derived clones can be compared to ensure no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5′ RACE RT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner. In one approach, a consensus primer set is designed for amplification of VL regions using a 5′ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3′ primer specific for the Ck region downstream of the V-J joining region. In a second approach, 5′RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions. Exemplary sequences encoding human constant regions include SEQ ID NO:61, which encodes a human IgG1 constant region, and SEQ ID NO:62, which encodes a human kappa light chain constant region.

In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hγ1 for the heavy chain and pCMV-Mc1 for the light chain. These vectors encode human γ1 and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. Following sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and assayed by western blot analysis for antibody production or ELISA for antigen binding. The chimeric antibodies are humanized as described above.

Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0. The cells can be nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489, U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for growth productivity and product quality in serum-free media. Top-producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., U.S. Pat. No. 5,786,464, U.S. Pat. No. 6,114,148, U.S. Pat. No. 6,063,598, U.S. Pat. No. 7,569,339, WO2004/050884, WO2008/012142, WO2008/012142, WO2005/019442, WO2008/107388, and WO2009/027471, and U.S. Pat. No. 5,888,809).

H. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavy and light chains described above (e.g., SEQ ID NOS:63-64, 67-68, 71-73, and 77-80). SEQ ID NOS:92-93, 99-100, and 102 are additional examples of nucleic acids encoding heavy and light chains described above. Typically, the nucleic acids also encode a signal peptide fused to the mature heavy and light chains (e.g., signal peptides having amino acid sequences of SEQ ID NOS:18, 28, 39, and 40 (heavy chain) and 19, 29, and 41 (light chain) that can be encoded by SEQ ID NOS:65, 69, 74, and 75, respectively (heavy chain) and 66, 70, and 76, respectively (light chain)). Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector.

I. Conjugated Antibodies

Antibodies that specifically bind to antigens, such as the LG4-5 modules of the G domain of laminin α4, that are preferentially present in cancers and tumors are useful in targeting cancer or tumor cells for destruction. Likewise, antibodies that specifically bind the LG4-5 modules of the G domain of laminin α4 can be useful in targeting cells involved in autoimmune diseases or any other diseases mediated at least in part by expression of the LG4-5 modules of the G domain of laminin α4. For example, such antibodies can be conjugated with other therapeutic or cytotoxic agents. See WO 03/057838. Likewise, such antibodies can be conjugated with other proteins, other antibodies, and/or detectable labels. See WO 03/057838; U.S. Pat. No. 8,455,622. Such therapeutic agents can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a patient. Examples of such conditions or diseases include cancers and autoimmune diseases. Therapeutic agents include cytotoxic agents, cytostatic agents, radiotherapeutic agents, immunomodulators, or any biologically active agents that facilitate or enhance the activity of the antibody. Such cytotoxic agents can be any agent that is toxic to a cell. A cytostatic agent can be any agent that inhibits cell proliferation. An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response. A radiotherapeutic agent can be any molecule or compound that emits radiation. If such therapeutic or cytotoxic agents are coupled to a tumor-specific antibody, such as the antibodies described herein, the coupled therapeutic or cytotoxic agents will have a specific affinity for tumor cells or cancer cells over normal cells. Likewise, the coupled therapeutic or cytotoxic agents will have a specific affinity for laminin-α4-expressing cells over other cells. Consequently, administration of the conjugated antibodies directly targets cancer cells with minimal damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic or cytotoxic agents that are too toxic to be administered on their own. In addition, smaller quantities of the therapeutic or cytotoxic agents can be used.

Antibodies can be modified to act as immunotoxins. See, e.g., U.S. Pat. No. 5,194,594. For example, ricin, a cellular toxin derived from plants, can be coupled to antibodies by using the bifunctional reagents S-acetylmercaptosuccinic anhydride for the antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). The coupling results in loss of B-chain binding activity of ricin, while impairing neither the toxic potential of the A-chain of ricin nor the activity of the antibody. Similarly, saporin, an inhibitor of ribosomal assembly, can be coupled to antibodies via a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

Radioisotopes can also be linked to antibodies, such as, for example, yttrium⁹⁰ (90Y), indium¹¹¹ (111In), ¹³¹I, ⁹⁹mTc, radiosilver-111, radiosilver-199, and Bismuth²¹³. Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates. For radiosilver-11 and radiosilver-199 linkage, sulfur-based linkers may be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

Other therapeutic agents may also be linked to antibodies. Therapeutic agents are usually cytotoxic or cytostatic. For example, antibodies can be conjugated with toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors, such as tubulin binding agents (e.g., auristatins), or minor groove binding agents, such as calicheamicin. Other representative therapeutic agents include agents known to be useful for treatment, management, or amelioration of a cancer or an autoimmune disease or symptoms of a cancer or an autoimmune disease. Examples of such therapeutic agents are disclosed elsewhere herein.

Antibodies can also be coupled with other proteins. For example, antibodies can be coupled with Fynomers. Fynomers are small binding proteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble, and they can lack cysteine residues and disulfide bonds. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for creating multi-specific fusion proteins based on antibodies. For example, Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to create bi- and tri-specific FynomAbs with different architectures. Fynomers can be selected using Fynomer libraries through screening technologies using FACS, Biacore, and cell-based assays that allow efficient selection of Fynomers with optimal properties. Examples of Fynomers are disclosed in Grabulovski et al., J. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013); and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to one or more other antibodies (e.g., to form antibody heteroconjugates). Such other antibodies can bind to different epitopes within the LG4-5 modules of the G domain of laminin α4, or can bind to a different target antigen.

Antibodies can also be coupled with a detectable label. Such antibodies can be used, for example, for diagnosing a cancer or an autoimmune disease, for monitoring progression of a cancer or an autoimmune disease, and/or for assessing efficacy of treatment. Such antibodies can be useful for performing such determinations in subjects having or being susceptible to a cancer or an autoimmune disease, or in appropriate biological samples obtained from such subjects. Representative detectable labels that may be coupled or linked to an antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin/biotin and avidin/biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-111, radiosilver-199, Bismuth²¹³, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin; positron emitting metals using various positron emission tomographies; nonradioactive paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes.

Therapeutic agents, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate (e.g., a linker), to a murine, chimeric, veneered, or humanized antibody using techniques known in the art. See e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers that release the drugs under acidic or reducing conditions or on exposure to specific proteases can be employed. Likewise, different linkers that release the coupled therapeutic agents, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed.

V. Therapeutic Applications

The above antibodies can be used for treating or effecting prophylaxis of a disease in a patient having or at risk for the disease mediated at least in part by expression of the LG4-5 modules of the G domain of laminin α4. In some such diseases, the LG4-5 modules of the G domain of laminin α4 contribute to progression of the disease by means of interaction with a heparan sulfate proteoglycan, such as syndecan-1, syndecan-2, syndecan-3, or syndecan-4.

Such a disease can be a cancer. Exemplary cancers to be treated have been described above. The methods are particularly amendable to treatment of a cancer showing a detectable level of the LG4-5 modules of the G domain of laminin α4 (e.g., by immunoassay using one of the disclosed antibodies). Some such cancers show an increased expression of laminin α4 compared with noncancerous tissue of the same type. Some such cancers are those that have been reported to be associated with laminin α4 expression, including glioma and glial tumors (see, e.g., Nagato et al., Int. J. Cancer 117:41-50 (2005); Ljubimova et al., Cancer 101(3):604-612 (2004)), hepatocellular carcinoma (see, e.g., Huang et al., J Cancer Res. Clin. Oncol. 134(6):705-14 (2008)), renal cell carcinoma (see, e.g., Vainionpaa et al., Lab Invest. 87(8):780-791 (2007)), oral squamous cell carcinoma (see, e.g., Takkunen et al., Histochem. Cell Biol. 130(3):509-525 (2008)), prostate cancer (see, e.g., Sprenger et al., Neoplasia 10(12):1350-1361 (2008)), and melanoma (see, e.g., Lugassy et al., J. Cutan. Pathol. 36(12):1237-1243 (2009)). Some such cancers are those associated with expression of one or more syndecans, such as breast cancer, cancer of the uterine cervix, colorectal cancer, endometrial cancer, fibrosarcoma, gallbladder cancer, gastric cancer, glioma, head and neck cancer, hepatocellular carcinoma, juvenile nasopharyngeal angiofibroma, lung cancer, melanoma, mesothelioma, myeloma, neuroendocrine tumors, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, and prostate cancer. See, e.g., Theocharis et al., FEBS Journal 277:3904-3923 (2010). Some of the above antibodies are useful for the treatment of melanoma, breast cancer, lung cancer, and colorectal cancer. Some antibodies may be useful for the treatment of uterine cancer, cervical cancer, endometrial cancer, fibrosarcoma, gallbladder cancer, gastric cancer, glioma, head and neck cancer, hepatocelleular carcinoma, juvenile nasopharyngeal angiofibroma, mesothelioma, myeloma, neuroendocrine, tumors, oral cancer, squamous cell carcinoma, oral squamous cell carcinoma, osteosarcoma, pancreatic cancer and/or prostate cancer. Some antibodies may be useful in treating metastatic tumors. In some instances the patient has a brain cancer or another type of CNS or intracranial tumor. For example, the patient can have an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglial tumor (e.g., oligodendroglioma, anaplastic oligodendroglioma), ependymal cell tumor (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixed glioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma), neuroepithelial tumor of uncertain origin (e.g., polar spongioblastoma, astroblastoma, gliomatosis cerebri), tumor of the choroid plexus (e.g., choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixed neuronal-glial tumor (e.g., gangliocytoma, dyplastic gangliocytoma of cerebellum, ganglioglioma, anaplastic ganglioglioma, desmoplastic infantile ganglioma, central neurocytoma, dysembryoplastic neuroepithelial tumor, olfactory neuroblastoma), pineal parenchyma tumor (e.g., pineocytoma, pineoblastoma, mixed pineocytoma/pineoblastoma), or tumor with mixed neuroblastic or glioblastic elements (e.g., medulloepithelioma, medulloblastoma, neuroblastoma, retinoblastoma, ependymoblastoma). Some antibodies may be useful in treating other cancers.

Such a disease can be an autoimmune disease. Autoimmune diseases include systemic autoimmune diseases, organ- or tissue-specific autoimmune diseases, and diseases that exhibit autoimmune-type expressions. In these diseases, the body develops a cellular and/or humoral immune response against one of its own antigens, leading to destruction of that antigen and potentially crippling and/or fatal consequences. The cellular response if present can be B-cell or T-cell or both. TH17 cells, a lineage T helper cells characterized by production of interleukin (IL)-17 and IL-22, have been reported to enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in mice. See, e.g., Cua et al., Nature 421: 744-748 (2003); Ivonov et al., Cell 126: 1121-1133 (2006). TH17 cells may initiate or propagate an inflammatory response by their specific recruitment to and infiltration of tissue.

Examples of autoimmune diseases include Graves' disease, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus (type 1 diabetes), insulin-resistant diabetes mellitus (type 2 diabetes), immune-mediated infertility, autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune thrombocytopenic purpura, pernicious anemia, myasthenia gravis, Guillain-Barre syndrome, stiff man syndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture's syndrome, autoimmune uveitis, temporal arteritis, Bechet's disease, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, autoimmune hepatitis, autoimmune oophoritis, fibromyalgia, polymyositis, dermatomyostis, ankylosing spondylitis, Takayashu arteritis, panniculitis, pemphigoid, vasculitis of unknown origin, anca negative vasculitis, anca positive vasculitis, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, scleroderma, systemic necrotizing vasculitis, Wegener's granulomatosis, CREST syndrome, antiphospholipid syndrome, Sjogren's syndrome, eosinophilic gastroenteritis, atypical topical dermatitis, cardiomyopathy, post-infectious syndromes, postinfectious endomyocarditis, celiac disease, multiple sclerosis, sarcoidosis, and psoriasis

Although an understanding of mechanism is not required for practice, it is believed that any or all of the following mechanisms may contribute to treatment of cancer. The antibodies may treat the cancer by inhibiting tumor cell adhesion, inhibiting laminin-α4-mediated signaling events, or inhibiting interaction of laminin α4 with a syndecan. The antibodies may additionally or alternatively treat cancer by inducing processing or clearance of the LG4-5 modules of the G domain of laminin α4. The antibodies may additionally or alternatively inhibit metastasis or cancer cell invasion. Binding of antibodies to LG4-5 modules of the G domain of laminin α4 may also affect cell adhesion, signaling mechanisms involved in cell proliferation, growth, resisting cell death, angiogenesis, or other characteristics of cancers. The antibodies may inhibit tumor growth via inhibiting Akt activation and subsequent cell survival/proliferation signaling. In some instances, the antibodies disrupt or inhibit angiogenesis by altering endothelial D114/Notch signaling. In some cases, the disruption or inhibition of angiogenesis by the antibodies involves disrupting the interaction between laminin α4 and integrins, such as integrins comprising integrin α2, integrin α6, or integrin β1. Antibody-drug conjugates have additional mechanisms of action including the cytotoxic or cytostatic effect of the linked agent, typically after uptake within a cancer cell. Antibody-drug conjugates can also act by such mechanisms of action in other targeted cells. Antibody-drug conjugates may also induce tumor-associated macrophage toxicity.

Other diseases treatable by antibodies of the invention include obesity and obesity-related diseases, such as obesity-related orphan diseases. Obesity is a disease caused by excessive food energy intake, lack of physical activity, and/or genetic susceptibility. A body mass index (BMI)>35 indicates severe obesity, a BMI>40 indicates morbid obesity, and a BMI>45 indicates super obesity. Obesity-related diseases include diseases and disorders that are associated with, are caused by, or result from obesity. Examples of obesity-related diseases include cardiovascular diseases, type 2 diabetes, sleep apnea, cancer, osteoarthritis, asthma, fatty liver, and non-alcoholic steatohepatitis (NASH).

NASH is characterized by hepatic inflammation and fat accumulation. The primary risk factors are obesity, diabetes, and dyslipidemia. There is a strong link with cirrhosis and hepatocarcinoma. NASH is associated with elevated AST/ALT (ratio of concentration of aspartate transaminase (AST) and alanine transaminase (ALT)), often without symptoms. Treatments for NASH include lifestyle changes (diet and exercise), bariatric surgery, and pharmaceuticals with mechanisms including absorption reduction (Xenical/Alli (lipase inhibitor)), appetite suppression (Belviq, Byetta, Symlin, Qsymia), and metabolic stimulation (Beloranib).

Examples of obesity-related orphan diseases include Prader-Willi syndrome (e.g., with hyperphagia), craniopharyngioma (e.g., with hyperphagia), Bardet-Biedl syndrome, Cohen syndrome, and MOMO syndrome. Prader-Willi syndrome is a rare genetic disease caused by gene deletion/silencing on chromosome 15. The symptoms include neurocognitive symptoms (intellectual disability, autistic behaviors, uncontrolled appetite (hypothalamic)), slow metabolism, and endocrine disorders (e.g., growth hormone deficiency (GHD), adrenal deficiency (AD)).

Antibodies are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated (e.g., cancer). If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.

Exemplary dosages for an antibody are 0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mg as a fixed dosage. The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some antibodies can be administered into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can be, for example, by infusion over a period such as 30-90 min.

The frequency of administration depends on the half-life of the antibody in the circulation, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.

The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the patient.

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Treatment with antibodies described herein can be combined with other treatments effective against the disorder being treated. When used in treating cancer, the antibodies can be combined with chemotherapy, radiation, stem cell treatment, surgery, or treatment with other biologics including Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or antibodies to the EGF receptor, such as (Erbitux®, cetuximab), and Vectibix® (panitumumab). Chemotherapy agents include chlorambucil, cyclophosphamide or melphalan, carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine and vinblastine.

VI. Other Applications

The antibodies can be used for detecting laminin α4 in the context of clinical diagnosis or treatment or in research. More specifically, the antibodies can also be used for detecting the LG4-5 modules of the G domain of laminin α4, or fragments thereof, in the context of clinical diagnosis or treatment or in research. For example, the antibodies can be used to detect the presence of the LG4-5 modules of the G domain of laminin α4 in a biological sample as an indication that the biological sample comprises cancer cells or tumor cells. Binding of the antibodies to the biological sample can be compared to binding of the antibodies to a control sample. The control sample and the biological sample can comprise cells of the same tissue origin. In some assays, the cancer cells that may be present in the biological sample arose from the same cell type as the type of cells in the control sample. Control samples and biological samples can be obtained from the same individual or different individuals and on the same occasion or on different occasions. If desired, multiple biological samples and multiple control samples are evaluated on multiple occasions to protect against random variation independent of the differences between the samples. A direct comparison can then be made between the biological sample(s) and the control sample(s) to determine whether antibody binding (i.e., the presence of the LG4-5 modules of the G domain of laminin α4) to the biological sample(s) is increased, decreased, or the same relative to antibody binding to the control sample(s). Increased binding of the antibody to the biological sample(s) relative to the control sample(s) indicates the presence of cancer in the biological sample(s). In some instances, the increased antibody binding is statistically significant. Optionally, antibody binding to the biological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher than antibody binding to the control sample.

In addition, the antibodies can be used to detect the presence of the LG4-5 modules of the G domain of laminin α4 in a biological sample to monitor and evaluate the efficacy of a therapeutic agent being used to treat a patient diagnosed with cancer. A biological sample from a patient diagnosed with cancer is evaluated to establish a baseline for the binding of the antibodies to the sample (i.e., a baseline for the presence of the LG4-5 modules of the G domain of laminin α4 in the sample) before commencing therapy with the therapeutic agent. In some instances, multiple biological samples from the patient are evaluated on multiple occasions to establish both a baseline and measure of random variation independent of treatment. A therapeutic agent is then administered in a regime. The regime may include multiple administrations of the agent over a period of time. Optionally, binding of the antibodies (i.e., presence of the LG4-5 modules of the G domain of laminin α4) is evaluated on multiple occasions in multiple biological samples from the patient, both to establish a measure of random variation and to show a trend in response to immunotherapy. The various assessments of antibody binding to the biological samples are then compared. If only two assessments are made, a direct comparison can be made between the two assessments to determine whether antibody binding (i.e., presence of the LG4-5 modules of the G domain of laminin α4) has increased, decreased, or remained the same between the two assessments. If more than two measurements are made, the measurements can be analyzed as a time course starting before treatment with the therapeutic agent and proceeding through the course of therapy. In patients for whom antibody binding to biological samples has decreased (i.e., the presence of the LG4-5 modules of the G domain of laminin α4 has decreased), it can be concluded that the therapeutic agent was effective in treating the cancer in the patient. Preferably, the decrease in antibody binding is statistically significant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Assessment of antibody binding can be made in conjunction with assessing other signs and symptoms of cancer.

The antibodies can also be used as research reagents for laboratory research in detecting laminin α4, or more specifically, the LG4-5 modules, or fragments thereof, of the G domain of laminin α4. In such uses, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of kit with all the necessary reagents to perform the assay for laminin α4, or more specifically, the LG4-5 modules of the G domain of laminin α4, or fragments thereof. The antibodies can also be used to purify laminin α4, laminins containing laminin α4, or binding partners of laminin α4, e.g., by affinity chromatography.

The antibodies can also be used for inhibiting cell adhesion in a biological sample. Preferably, the cell adhesion is dependent on laminin α4. For example, the cell adhesion is mediated by the LG4-5 modules of the G domain of laminin α4. The biological sample can comprises a tumor, cancer, or cells derived therefrom. Optionally, the tumor or cancer is melanoma. An exemplary cell adhesion assay is described in the examples. In some instances, cell adhesion is inhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75% or 30%-70%)

The antibodies can also be used for inhibiting binding of laminin α4 to a syndecan in a biological sample. Optionally, the syndecan is syndecan-1, syndecan-2, syndecan-3, or syndecan-4. Inhibition may be demonstrated in a binding assay assessing the ability of syndecan-expressing cells to bind laminin α4 in the presence or absence of the antibodies. Optionally, inhibition of a test antibody can be demonstrated in comparison to an irrelevant control antibody not binding to the LG4-5 modules of the G domain of laminin α4 or in comparison to vehicle lacking any antibody. For example, binding of laminin α4 to the syndecan is inhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75% or 30%-70%).

The antibodies can also be used for inhibiting laminin-α4-induced pAkt activation in a biological sample. An exemplary assay is described in the examples. In some methods, laminin-α4-induced pAkt activation is inhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g., 10%-75% or 30%-70%).

All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Examples Example 1 Identification of LG4-5-Domain-Specific Anti-LAMA4 Monoclonal Antibodies

LAMA4 tumor expression has been reported to correlate with tumor grade, recurrence, and survival, while downregulation has been reported to inhibit tumor invasion in vitro and in vivo. See, e.g., Ljubimova et al., Cancer 101: 604-612 (2004) and Nagato et al., Int J Cancer 117: 41-50 (2005). Interestingly, the LG4-5 modules of the G domain of LAMA4 homologue LAMA3 have been reported to be exclusively found in squamous cell carcinomas (SCC) but absent/processed in normal healthy tissue. Furthermore, the LG4-5 modules of the G domain of LAMA3 have been reported to be necessary for SCC tumor growth, as a polyclonal antibody has been reported to inhibit tumor growth in a SCC xenograft mouse model. See, e.g., Tran et al., Cancer Res. 68: 2885-2894 (2008). It was thus of great interest to determine (1) if LAMA4 is processed in a similar fashion in tumors and (2) whether an LG4-5-specific anti-LAMA4 antibody could be efficacious in LAMA4-positive tumors.

Monoclonal antibodies against the LG4-5 modules of the G domain of LAMA4 were generated as described in Materials and Methods. The specific binding between the monoclonal antibodies and the LG4-5 modules of the G domain of LAMA4 was confirmed by assessing the monoclonal antibodies' ability to stain LAMA4-fragment-displaying human embryonic kidney 293 cells by flow cytometry. Fluorescent signal was assessed via flow cytometric analyses and plotted as mean fluorescence intensity (MFI) as shown in FIG. 1. A LG1-3-specific LAMA4 antibody was able to bind 293 cells displaying LG1-5 and LG1-3, but not LG4-5 of the LAMA4 protein. Conversely, clones 6C12, 13G10, and 15F7 were able to specifically bind 293 cells displaying LG1-5 and LG4-5, but not LG1-3.

Relative binding and on/off rates for the 15F7, 6C12, and 13G10 antibodies were analyzed by ForteBio as shown in FIG. 2A-C, respectively. Antibody concentrations were kept constant at 100 nM, and the concentration of LAMA4 was varied as indicated in FIG. 2A-C. For each concentration of LAMA4, two lines are presented in FIG. 2A-C: a bolded line representing the raw data and a non-bolded line representing the statistical fitting of the raw data. Detailed binding kinetic parameters (association rate (k_(a)), dissociation rate (k_(d)), and binding affinity constant (K_(D))) were determined by Biacore for 13G10, 15F7, and 6C12, as shown in Table 1. The 15F7 antibody displayed the highest binding magnitude and slowest off rates in the ForteBio and Biacore assays.

TABLE 1 Biacore Assay Comparing Binding of 15F7, 13G10, and 6C12 to LAMA4 Human LAMA4 Murine LAMA4 Antibody k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 13G10 3.02 × 10⁵  7.4 × 10⁻³ 2.44 × 10⁻⁸  3.78 × 10⁵ 4.49 × 10⁻³ 1.19 × 10⁻⁸ 15F7 1.14 × 10⁶ 7.81 × 10⁻⁴ 6.84 × 10⁻¹⁰ 1.05 × 10⁶ 6.58 × 10⁻⁴ 6.27 10⁻¹⁰ 6C12 3.28 × 10⁵ 1.02 × 10⁻³ 3.11 × 10⁻⁹  4.69 × 10⁵ 1.11 × 10⁻³ 2.38 × 10⁻⁹

The 6C12, 13G10, and 15F7 antibodies, along with mouse IgG control, were tested for their ability to bind LAMA4-displaying cells. To test LAMA4 binding capacity, serially diluted antibodies were pre-incubated with LAMA4-displaying human 293 cells, followed by anti-human-650 secondary antibody incubation as described in the Materials and Methods. Binding capacity was assessed by flow cytometry as shown in FIG. 3. 15F7 again displayed the highest binding magnitude.

Competition experiments were carried out to differentiate the 6C12, 13G10, and 15F7 antibodies by epitope binding. Binding of the antibodies to LAMA4-displaying 293 cells was assessed using decreasing ratios (5:1, 1:1, and 1:5) of blocking antibody to binding antibody, with mouse IgG1 being used as a negative control. Binding of the 6C12, 13G10, and 15F7 antibodies was assessed by flow cytometery as shown in FIG. 4A-C, respectively. Fluorescent signal was assessed via flow cytometric analyses and plotted as mean fluorescence intensity (MFI). All three antibodies were able to compete with each other for LG4-5 binding, with each being having higher blocking efficacy at the 5:1 ratio ((blocking antibody):(binding antibody)) and lower blocking efficacies as the ratio decreases. These results indicate that these antibodies all bind similar epitopes on the LAMA4 protein.

Example 2 LG4-5-Domain-Specific Anti-LAMA4 Monoclonal Antibody Exclusively Stains Human and Mouse Melanoma Tissue

To determine whether the LG4-5 modules of the G domain of LAMA4 are exclusively found in tumor tissue, WM-266-4 human melanoma cells were tested for LAMA4 expression with polyclonal anti-LAMA4 antibodies. Staining was undertaken for both MCAM and LAMA4. MCAM-positive WM-266-4 cells were positive for LAMA4 expression.

WM-266-4 tumors were implanted subcutaneously in nude mice and allowed to grow for five weeks. Following transcardiac perfusion with PBS, tumors and healthy tissue were frozen, sectioned, and stained with mouse IgG control, LG1-3-specific, and LG4-5-specific monoclonal antibodies 6C12, 13G10, and 15F7 as described in Materials and Methods. LG1-3-specific and LG4-5-specific antibodies were both able to stain WM-266-4 subcutaneous tumors and vasculature.

Additional subcutaneous tumor experiments were performed using a mouse melanoma cell line, B16. B16 mouse melanoma cells were subcutaneously injected or intravenously injected into mice and allowed to metastasize to the lungs. Staining of B16 tumor tissue and healthy mouse brain, liver, kidney, and lung tissues was undertaken with a LG1-3-specific antibody, with mouse IgG1 used as a control. The LG1-3-specific antibody was able to robustly stain all tissue types tested. Staining of B16 tumor tissue and healthy mouse brain, liver, kidney, and lung tissues was also undertaken with the LG4-5-specific antibodies, 6C12, 13G10, and 15F7. In contrast to the LG1-3-specific antibody, these three LG4-5-specific antibodies exclusively stained the B16 tumor vasculature in the primary subcutaneous tumors and were not able to robustly stain any of the healthy tissues. Staining of a sample containing B16 lung metastatic foci and healthy adjacent lung tissue was undertaken with the LG1-3-specific antibody, the LG4-5-specific antibodies (6C12, 13G10, and 15F7), and a control mouse IgG1. The LG1-3-specific antibody was able to robustly stain both the metastatic foci and the healthy adjacent tissue. In contrast, the 6C12, 13G10, and 15F7 antibodies were not able to stain the healthy adjacent lung tissue and exclusively stained B16 tumor vasculature in metastatic foci.

Samples of healthy human skin and skin melanoma from two distinct patients were stained with a LG4-5-specific antibody, 15F7. Consistent with the staining results described above, 15F7 was able to stain both skin melanoma samples but was unable to stain the healthy skin tissue.

Other human tumor types were stained as well. As shown in FIG. 5A-C, a LG-4-5-specific antibody, 15F7, was used to stain tumor microarray slides for human breast, colon, and lung tumors, respectively. Normal tissue controls are enclosed in the white rectangles. Consistent with the staining of the melanomas described above, 15F7 preferentially stained the majority of tumor patient samples in the tumor microarray slides for human breast, colon, and lung cancers. These results are consistent with a model whereby the LG4-5 modules of the G domain of LAMA4 are exclusively found in various human and mouse tumor types, indicating that the LG4-5 modules of the G domain of LAMA4 can be a tumor-specific marker for diagnostics and targeted tumor therapeutics.

Example 3 LG4-5-Domain-Specific Anti-LAMA4 Monoclonal Antibody Inhibits Human Melanoma Cell Adhesion and Drives Antibody-Drug Conjugate Cell Toxicity

To determine the functional consequences of targeting the LG4-5 modules of the G domain of LAMA4 with anti-LG4-5 antibodies, recombinant LG4-5-coated ELISA plates were incubated with 20 ug/ml 15F7, 6C12, and 13G10 (or mouse IgG1 control) and were then assayed for their ability to bind human melanoma cell line WM-266-4 as described in Materials and Methods. Buffer was used as a negative control and heparin was used as a positive control. A sample with no cells was used as an additional negative control. The results of the cell adhesion assay are shown in FIG. 6. The results are presented in arbitrary units (A.U.) on the y-axis. Although mouse IgG1 control failed to block LAMA4-mediated cell adhesion, all three anti-LG4-5 antibodies were able to inhibit LAMA4-mediated human melanoma cell adhesion by approximately 35% or greater, indicating that anti-LG4-5 antibodies can block cell adhesion events necessary for tumor cell adhesion, proliferation, and metastasis.

Because the LG4-5 domain of LAMA4 is highly enriched in various human tumor tissues when compared to healthy human tissue, we tested anti-LG4-5 antibodies as candidates for antibody-drug conjugate targeting strategies. As shown in FIG. 7, LAMA4+WM-266-4 cells were incubated with the 15F7 anti-LG4-5 antibody or a mouse isotype control along with ribosomal toxin saporin-conjugated anti-mouse secondary. Although LAMA4 LG4-5 is a soluble extracellular matrix protein, 15F7 was able to strongly mediate saporin-mediated cell toxicity.

These data indicate that anti-LG4-5 antibodies block WM-266-4 human melanoma cell adhesion and are strong candidates for antibody-drug conjugate therapeutic approaches. Targeting LG4-5 could be efficacious in slowing tumor growth and metastasis.

Example 4 Design of Humanized 15F7 Antibodies

The starting point or donor antibody for humanization was the mouse antibody 15F7. The heavy chain variable amino acid sequence of mature m15F7 is provided as SEQ ID NO:16. The light chain variable amino acid sequence of mature m15F7 is provided as SEQ ID NO:17. The heavy chain CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:20, 21, and 22, respectively (as defined by Kabat). The light chain CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:23, 24, and 25, respectively (as defined by Kabat). Kabat numbering is used throughout in this Example.

The variable kappa (Vk) of m15F7 belongs to mouse Kabat subgroup 5, which corresponds to human Kabat subgroup 1. The variable heavy (Vh) of m15F7 belongs to mouse Kabat subgroup 5a, which corresponds to Kabat subgroup 1. See Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242, 1991. The 11-residue CDR-L1 belongs to canonical class 1, the 7-residue CDR-L2 belongs to canonical class1, and the 9-residue CDR-L3 belongs to canonical class 1 in Vk. See Martin & Thornton, J. Mol. Biol. 263:800-15, 1996. The 5-residue CDR-H1 (as defined by Kabat) belongs to canonical class 1, and the 17-residue CDR-H2 belongs to canonical class 1. See Martin & Thornton, J Mol. Biol. 263:800-15, 1996. The CDR-H3 has no canonical classes, but the 10-residue loop probably has a kinked base according to the rules of Shirai et al., FEBS Lett. 455:188-97 (1999).

The residues at the interface between the Vk and Vh domains are usual.

A search was made over the protein sequences in the PDB database (Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to find structures which would provide a rough structural model of 15F7. The crystal structure of the antibody against monocyte chemoattractant proteins (MCPs) was used for Vk structure. It retains the same canonical structure for the loop as 15F7 (pdb code 2BDN, resolution 2.53A). The heavy chain of the antibody against LeuT mutants (pdb code 3TT1, resolution 3.1A) was used for Vh structure. It contains the same canonical structures for CDR-H1 and CDR-H2 as that of 15F7 VH, and also the same length CDR-H3 with a kinked base. BioLuminate was used to model a rough structure of 15F7 Fv.

A search of the non-redundant protein sequence database from NCBI with CDR“X”ed 15F7 Fv allowed selection of suitable human frameworks into which to graft the murine CDRs. For Vh, two human Ig heavy chains, ACF36857.1 and BAC01530.1 (SEQ ID NOS:96 and 97, respectively) were chosen. They share the canonical form of 15F7 CDR-H1 and H2, and H3 of BAC01530.1 is 10 residues long with a predicted kinked base. For Vk, two human kappa light chains, having NCBI accession codes AAY33350.1 and BAC01583.1 (SEQ ID NOS:53 and 98, respectively), were chosen. They have the same canonical classes for CDR-L1, L2, and L3 as that for the parental Vk. Humanized 15F7 heavy and light chain variable region sequences having no backmutations or other mutations are provided as SEQ ID NOS:55 and 56.

Two humanized heavy chain variable region variants and two humanized light chain variable region variants were constructed containing different permutations of substitutions (Hu15F7VHv1-2 (SEQ ID NOS:57 and 58, respectively) and Hu15F7VLv1-2 (SEQ ID NOS:59 and 60, respectively)) (Tables 2-5). The exemplary humanized Vh and Vk designs, with backmutations and other mutations based on selected human frameworks, are shown in Tables 2 and 3, respectively. The gray-shaded areas in the first column in Tables 2 and 3 indicate the CDRs as defined by Chothia, and the gray-shaded areas in the remaining columns in Tables 2 and 3 indicate the CDRs as defined by Kabat. SEQ ID NOS:57-60 contain backmutations and other mutations as shown in Table 4. The amino acids at positions H1, H20, H27, H30, H38, H40, H41, H48, H66, H67, H69, H75, H82A, H91, L8, L42, L45, L49, L69, and L80 in Hu15F7VHv1-2 and Hu15F7VLv1-2 are listed in Table 5. The amino acids at position L104 in Hu15F7VLv1-2 are also listed in Table 5.

TABLE 2 Humanized 15F7 Vh Regions

TABLE 3 Humanized 15F7 Vk Regions

TABLE 4 V_(H), V_(L) Backmutations and Other Mutations V_(L) Exon Donor Framework V_(L) Variant Acceptor Sequence Residues Hu15F7VLv1 NCBI accession codes L8, L42, L45, L49, L69, (SEQ ID NO: 59) AAY33350.1 and L80 BAC01583.1 (SEQ ID NOS: 53 and 98) Hu15F7VLv2 NCBI accession codes L42, L49, L69, L104 (SEQ ID NO: 60) AAY33350.1 and BAC01583.1 (SEQ ID NOS: 53 and 98) Hu15F7VHv1 NCBI accession codes H1, H20, H27, H30, (SEQ ID NO: 57) ACF36857.1 and H38, H40, H41, H48, BAC01530.1 H66, H67, H69, H75, (SEQ ID NOS: 96 and 97) H82A, H91 Hu15F7VHv2 NCBI accession codes H1, H20, H27, H30, (SEQ ID NO: 58) ACF36857.1 and H38, H40, H48, H66, BAC01530.1 H67, H75, H82A, H91 (SEQ ID NOS: 96 and 97)

TABLE 5 Kabat Numbering of Framework Residues for Backmutations and Other Mutations in Humanized 15F7 Antibodies AAY33350.1 BAC01583.1 ACF36857.1 BAC01530.1 light light heavy heavy Mouse Hu15F Hu15F Hu15F Hu15F Residue chain chain chain chain 15F7 7VL1 7VL2 7VH1 7VH2 L8 P P — — S S P — — L42 K K — — N N N — — L45 K K — — R R K — — L49 Y Y — — S S S — — L69 T T — — K K K — — L80 P P — — T T P — — L104 L L — — L L V — — H1 — — Q Q Q — — E E H20 — — V V L — — L L H27 — — G G Y — — Y Y H30 — — S S T — — T T H38 — — R R K — — K K H40 — — A A R — — R R H41 — — P P A — — A P H48 — — M M I — — I I H66 — — R R K — — K K H67 — — V V A — — A A H69 — — I I L — — L I H75 — — T T S — — S S H82A — — S S R — — R R H91 — — Y Y F — — F F

The rationales for selection of the above positions in the light chain variable region as candidates for substitution are as follows.

P8S: P is more frequent than S in the human IgG framework, but because proline cis-trans isomerization affects protein folding, P was tried in one version and S in the other version.

K42N: N contacts interface residue F91 in VH and is therefore critical for maintaining antibody structure.

K45R: R and K have similar frequency in the human IgG framework, so R was tried in one version and K in the other version.

Y49S: S contacts LCDR2 and is critical.

T69K: K contacts LCDR1 and is critical.

P80T: P is more frequent than T in the human IgG framework, but because proline cis-trans isomerization affects protein folding, P was tried in one version and T was tried in the other version.

L104V: L was tried in one version, and V was tried in the other version.

The rationales for selection of the above positions in the heavy chain variable region as candidates for substitution are as follows.

Q1E: This is a mutation but not a backmutation. Glutamate (E) conversion to pyroglutamate (pE) occurs more slowly than from glutamine (Q). Because of the loss of a primary amine in the glutamine to pE conversion, antibodies become more acidic. Incomplete conversion produces heterogeneity in the antibody that can be observed as multiple peaks using charge-based analytical methods. Heterogeneity differences may indicate a lack of process control.

V20L: L is more frequent than V in the human IgG framework.

G27Y: This residue is within HCDR1 as defined by Chothia, so Y was used to maintain binding ability.

S30T: This residue is within HCDR1 as defined by Chothia, so T was used to maintain binding ability.

R38K: K contacts two interface residues, L45 and W47, in VH and is therefore critical.

A40R: R is located at the end of the KQRAGQG (VH amino acids 38-44) loop, which supports the two interface residues, V37 and L45 in VH. It is important for maintaining antibody folding.

P41A: P is more frequent than A in the human IgG framework, but introduction of P into the framework may cause some conformational changes, so P was tried in one version and A was tried in the other version.

M48I: I contacts HCDR2 and interface residues V37 and L45 in VH.

R66K: K contacts HCDR2.

V67A: A contacts HCDR2.

169L: I is more frequent than L in the human IgG framework, but L contacts HCDR1 and HCDR2, so I was tried in one version and L was tried in the other version.

T75S: Both T and S are frequent in the human framework.

S(82A)R: R contacts HCDR2 and is critical.

Y91F: F is an interface residue and is important to support antibody folding.

The two humanized light chain variable region variants and two humanized heavy chain variable region variants are as follows:

Hu15F7VL version 1 (P8S, K42N, K45R, Y49S, T69K, and P80T backmutations in lowercase):

(SEQ ID NO: 59) DIQMTQSsSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGnAPrLLI sGATSLETGVPSRFSGSGSGkDYTLTISSLQtEDFATYYCQQYWSIPY TFGGGTKLEIKR.

Hu15F7VL version 2 (K42N, Y49S, and T69K backmutations and L104V mutation in lowercase):

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGnAPKLLI sGATSLETGVPSRFSGSGSGkDFTLTISSLQPEDFATYYCQQYWSIPY TFGGGTKvEIKR.

Hu15F7VH version 1 (Q1E mutation and V20L, G27Y, 530T, R38K, A40R, P41A, M48I, R66K, V67A, 169L, T75S, S(82A)R, and Y91F backmutations in lowercase):

(SEQ ID NO: 57) eVQLQQSGAEVKKPGSSVKlSCKASGyTFtSYGLSWVkQraGQGLEWi GEIFPRSGNTYYNEKFKGkaTlTADKSsSTAYMELrSLRSEDTAVYfC ARGVRSPGAMDYWGQGTLVTVSS.

Hu15F7VH version 2 (Q1E mutation and V20L, G27Y, 530T, R38K, A40R, M48I, R66K, V67A, T75S, S(82A)R, and Y91F backmutations in lowercase):

(SEQ ID NO: 58) eVQLQQSGAEVKKPGSSVKlSCKASGyTFtSYGLSWVkQrPGQGLEWi GEIFPRSGNTYYNEKFKGkaTITADKSsSTAYMELrSLRSEDTAVYfC ARGVRSPGAMDYWGQGTLVTVSS.

Example 5 Binding Kinetic Analysis of Humanized 15F7 Antibodies

Binding kinetics of humanized 15F7 antibodies comprising a heavy chain selected from Hu15F7VHv1-2 (H1 and H2) and a light chain selected from Hu15F7VLv1-2 (L1 and L2) were characterized.

Chimeric 15F7, H1L1, H1L2, H2L1, H2L2, and buffer alone were tested for their ability to bind to LAMA4-fragment-displaying cells. To test LAMA4 binding capacity, serially diluted antibodies were pre-incubated with human 293 cells displaying LG4-5, followed by anti-human-650 secondary antibody incubation as described in the Materials and Methods. Fluorescent signal was assessed via flow cytometric analyses and plotted as mean fluorescence intensity (MFI) as shown in FIG. 8. The serially diluted H1L1, H1L2, H2L1, and H2L2 antibodies each showed binding capacity that is comparable to chimeric 15F7.

The specific binding of the chimeric 15F7, HILL H1L2, H2L1, and H2L2 antibodies to the LG4-5 modules of the G domain of LAMA4 was tested by assessing the antibodies' ability to stain LAMA4-fragment-displaying 293 cells by flow cytometry, as shown in FIG. 9. Each of the antibodies was able to specifically bind 293 cells displaying LG1-5 and LG4-5, but not LG1-3.

Relative binding and on/off rates were analyzed by ForteBio, as shown in FIGS. 10A and 10B. In FIG. 10A, the anti-His sensor was loaded with 10 ug/ml of purified His-LAMA4 followed by loading of 5 ug/m1 of m15F7, chimeric 15F7, H1L1, H1L2, H2L1, and H2L2. Association and dissociation were analyzed. In FIG. 10B, the goat anti-human Fc sensor was loaded with m15F7, chimeric 15F7, H1L1, H1L2, H2L1, and H2L2 as indicated at 5 ug/m1 followed by loading of 10 ug/ml of His-LAMA4. Association and dissociation were analyzed. Relative binding and on/off rates were comparable among all antibodies tested, with H1L1 and H2L1 displaying relative binding that was the same or higher than that for chimeric 15F7 and m15F7.

Biacore full binding kinetic analysis of antibodies was then carried out. SPR analysis was performed as described in the Materials and Methods. Detailed binding kinetic parameters (association rate (k_(assoc)), dissociation rate (k_(dissoc)), and binding affinity constant (K_(d))) were determined for chimeric 15F7, humanized H1L1, and humanized H2L1. Binding kinetic parameters for the humanized 15F7 variants H1L1 and H2L1 were comparable to those for chimeric 15F7 (see Table 6).

TABLE 6 Biacore Assay Comparing Binding of HU15F7 Variants and Chimeric 15F7 to LAMA4 Antibody k_(assoc) (M⁻¹s⁻¹) k_(dissoc) (s⁻¹) K_(d) (M) Chimeric 1.50 × 10⁶ 1.51 × 10⁻⁴ 1.0 × 10⁻¹⁰ H1L1 1.50 × 10⁶ 1.83 × 10⁻⁴ 1.2 × 10⁻¹⁰ H2L1 1.56 × 10⁶ 2.05 × 10⁻⁴ 1.3 × 10⁻¹⁰

Example 6 Anti-Laminin Antibodies Inhibit Laminin-411-Induced pAkt Activation

WM266.4 human tumor melanoma cells were serum-starved for 24 h and then resuspended into serum-free cell culture media with 10 ug/ml laminin 411 (LAMA4 in complex with gamma1 and beta1 chains) and 20 ug/ml 15F7 or mIgG1 control antibody for 30 minutes. BSA protein was used as a control for laminin 411. Cells were then spun down and lysed for immunoblot analyses. pAkt and total Akt levels were assessed by immunoblot. Ratios of these levels (pAkt/Akt) are shown in FIGS. 11A & B. Each condition (mIgG1+laminin 411; 15F7+laminin 411; and mIgG1+BSA) was tested in triplicate. FIG. 11A shows the results for each individual sample, and FIG. 11B shows the averages and standard errors for each condition. As shown in FIG. 11B, laminin 411 induced pAkt signaling (i.e., higher pAkt/Akt ratio) compared to BSA control, and 15F7 partially blocked laminin-411-induced pAkt activation (˜50% inhibition).

Example 7 Effects of Laminin 411 and Anti-Laminin Antibodies on Notch Signaling

Because Notch ligand D114 transcription/translation requires integrin ligation and subsequent phospho-Akt signaling, anti-LAMA4 antibodies are tested for effects on Notch signaling. HUVEC, WM266.4, and RAW cells are resuspended in cell culture media with 10 ug/ml laminin-411 (LAMA4 in complex with gamma1 and beta1 chains) and 20 ug/ml 15F7 or isotype control antibody for 24 hrs. BSA protein is used as a control for laminin 411. Cells are spun down and lysed for immunoblot analyses for cleaved/activated Notch1, D114, MCAM, actin, pAkt, and Akt. In addition, qPCR analysis for Hey1, MCP-1 (monocyte chemoattractant in inflammation), MCAM, LAMA4, and GAPDH is undertaken.

Example 8 Effects of Anti-Laminin Antibodies in In Vivo Obesity Models

Because Akt signaling is important for Notch signaling, and Notch signaling encourages growth of adipocytes, antibodies against LAMA4 are tested in in vivo obesity models for effects on weight gain/loss and adipocyte metabolism and lipolysis. High-fat diet (HFD)-driven weight gain in mice is assessed in response to anti-laminin 411 antibodies. Wild-type C57BL/6 mice are fed a high-fat diet (e.g., rodent diet with 45% kcal % fat, such as product #D12451 from Research Diets, Inc.) ad libitum. Two experimental groups are tested: (1) mice treated with control Ig; and (2) mice treated with 15F7. There are ten mice in each group, and each mouse is treated with 10 mg/kg/week antibody for three to four months. Weight measurements are taken every two to four weeks.

To assess localization of LAMA4 to adipose tissue, anti-LAMA4 antibody (compared to isotype control antibody) is intravenously administered to mice. Staining is then undertaken to assess localization to adipose tissue.

Example 9 Materials and Methods LAMA4 Fragment Purification

His-tagged LAMA4 G-domain fragments were cloned by standard procedures and transiently expressed in 293 cells. Protein was purified using a nickel-NTA column.

LAMA4 Knockout Mouse

Lama4 null mice originally obtained from Dr. Karl Tryggvason (Karolinska University).

Generation of Recombinant MCAM-Fc Protein

MCAM-Fc was generated in house by fusing the extracellular domain of human or mouse MCAM to human IgG1 and produced/purified in CHO cells using standard techniques.

Antibody Generation

Recombinant mouse laminin 4 (Lama4) obtained from R&D Systems and 10 week old Lama4 null mice originally obtained from Dr. Karl Tryggvason (Karolinska University) were used to develop the antibodies. Purified laminin α4 (LAMA4) was suspended in RIBI adjuvant at 10 ug LAMA4/25 ul adjuvant. Mice were anesthetized with isoflurane and 3 mice were immunized into each rear footpad or rear hock with 5 ug Lama4 in RIBI adjuvant while two mice were immunized with 12.5 ug Lama4 in RIBI adjuvant into each rear footpad or rear hock with a 27 gauge needle. Mice were injected following the above procedure on days 0, 4, 12, 16 and 20. On day 24 animals are euthanized and the popiteal and inguinal lymph nodes are removed in a sterile hood. The nodes are dissociated and fused with SP2/0 using a modification of the Kohler and Milstein protocol that incorporates Electrofusion instead of PEG fusion. Fused cells are plated into 96 well plates and allowed to grow.

When cells reach half to three quarters confluence screening begins. Briefly, Costar RIA/EIA plates were coated with rabbit ant-His tag (Anaspec #29673) at 1 ug/mL, 50 uL/well, in PBS for 1 hour. Plates were then blocked with 250 ul/well of 1% BSA/PBS for 15 minutes and then removed. His-tagged Lama4 was added to the plates at 0.25 ug/mL, 50 uL/well for 1 hour, and then washed 2×. 75 uL of supernatant from fusion plates was added and incubated for 1 hour, plates were washed 2×. Goat-anti-mouse (Jackson #115-035-164) was added at 1:2000 dilution in 0.5% BSA/PBS/TBST for 1 hour, then washed 5×. Plates were developed with 50 ul/well TMB (SurModics #TMBW24) for 5 minutes, and stopped with 15 uL 2N H2504, and read at 450 nm. Wells with OD greater than 1.0 were selected for additional screening. Cells from wells found positive by the ELISA were grown up and frozen. Supernatants were provided for the additional screening described below. Cells from wells meeting certain criteria described below were cloned using the Clonepix FL and screened using setting recommended by the company to find single cell clones. These were expanded and the antibody purified from supernatants.

LAMA4 pDisplay Flow Cytometric Binding Assay

Human LAMA4 G-domains 1-5 and variants were cloned into pDisplay expression construct (Life Technologies) and transiently transfected into 293 cells using standard procedures. Anti-LAMA4 antibodies were incubated with cells for 30 min at 4° C. and followed by anti-mouse-650 for 30 minutes at 4° C. Cells were analyzed for anti-laminin binding by flow cytometry using standard procedures.

WM-266-4 Cell Transfection and Staining

Cultured WM-266-4 cells were transfected with full-length human MCAM-GFP fusion constructs and stained with anti-LAMA4 antibodies using standard procedures.

WM-266-4 Human Melanoma Subcutaneous Tumor Tissue

Cells were cultured and 5×10⁵ cells per animal were subcutaneously administered above the shoulder in nude mice. After five weeks, animals were transcardially perfused with PBS and tumors were excised and snap frozen.

B16 Mouse Melanoma Subcutaneous Tumor Tissue

Cells were cultured and 5×10⁵ cells per animal were subcutaneously administered above the shoulder in c57bl6 mice. After five weeks, animals were transcardially perfused with PBS and tumors were excised and snap frozen.

B16 Mouse Melanoma Lung Metastasis Lung Tissue

Cells were cultured and 5×10⁵ cells per animal were intravenously injected into nude/beige mice from Charles River (offsite at Caliper LifeSciences). After three weeks, animals were transcardially perfused with PBS, and lungs were excised and snap frozen.

Fluorescence Microscopy/Standard Immunofluorescent Methods

Mouse tissue was snap frozen in OCT and sectioned at 10 uM. Sections were fixed in cold acetone and stained with anti-LAMA4 antibody (R&D systems).

Human Melanoma Cell Adhesion Assay

Recombinant 10 ug/ml mLAMA4 (R&D systems), was used to coat 96-well plates overnight at 4° C. Following PBS washing steps, wells were blocked with 1% BSA/MEM for 1 hr at room temperature. 20 ug/ml anti-LAMA4 antibodies in 0.1% BSA/MEM were added to plates for 1 hour at room temperature. WM-266-4 cells were resuspended with EDTA, wash and resuspended at 300,000 cells/ml in 0.1%/MEM, followed by 10 minutes in the tissue culture incubator at 37° C. with the tube cap off. Following two washes with FACS buffer (1% FBS in PBS), cells were resuspended with 650-conjugated anti-pan-laminin antibody (1:1000; Novus Biologicals) and incubated for 20 min at 4° C., and washed again. Without removing antibody solutions, add cell suspension to well and incubate uncovered in tissue culture incubator for 1.5 hrs. Following a PBS wash step, cells were stained/fixed with glutaraldehyde/crystal violet solution prior to plate reader analysis at 570 nm.

Generation of Fab Fragments

Fab fragments of all antibodies were generated using the Fab Micro Preparation kit following manufacturer's directions (Pierce). Removal of liberated Fc and verification of intact final product were monitored by SDS-PAGE, and concentration was determined using the bicinchoninic acid assay (Pierce).

SPR Measurements of Affinity

SPR analysis was performed using a Biacore T200 to compare the binding of the different laminin antibodies. For Fab preparations, anti-6× His antibody (GE Life Sciences) was immobilized on sensor chip C1 via amine coupling, and human His-laminin-α4, mouse His-laminin-α4 (both from R & D Systems), and an unrelated 6× His-tagged protein (as a reaction control) were captured at a level to ensure maximum binding of 25 RU. Various concentrations of Fab preparations ranging from 300-0.41 nM were passed over the captured ligands in parallel at a flow rate of 50 ul/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA), for 240 s association and varying durations of dissociation. Data were double-referenced to both an irrelevant sensor not containing His-tagged ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Data was then analyzed using either a heterogeneous ligand model or a global 1:1 fit.

For whole IgG, anti-mouse antibody was immobilized on sensor chip C1 (lacking dextran chains) via amine coupling, and laminin mAbs were captured to a level to ensure a maximum binding of analyte of 50 RU. Various concentrations of analyte (recombinant human or mouse His-laminin-α4 fragment starting at Q826 (R&D Systems)) ranging from 31.25 nM to 0.122 nM were passed over the captured ligand at 30 ul/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA, except where described) for 180 s association/900 s dissociation. Data were double-referenced to both an irrelevant sensor not containing His-tagged ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Where possible, data were analyzed using a global 1:1 fit. If kinetic sensorgram curvature did not allow proper model-fitting, a steady-state approximation was performed and reported.

Saporin-Mediated WM-266-4 Cell Toxicity Assay

Per manufacturer's instructions (Advanced Targeting Systems).

Human Melanoma Tissue and Tumor Microarray Slides

Unfixed healthy and melanoma human skin slides were obtained from Origene. Acetone-fixed tumor microarray (TMA) slides were obtained from Biochain. 

What is claimed is:
 1. An antibody that specifically binds to an epitope within the LG4-5 modules of the G domain of laminin α4.
 2. The antibody of claim 1 that does not inhibit binding of laminin α4 to MCAM.
 3. The antibody of claim 1 that binds to an epitope within LG4.
 4. The antibody of claim 1 that binds to an epitope within LG5.
 5. The antibody of claim 1 that binds to an epitope to which both LG4 and LG4 contribute residues.
 6. The antibody of claim 1 that inhibits binding of laminin α4 to a heparan sulfate proteoglycan.
 7. The antibody of claim 6, wherein the heparan sulfate proteoglycan is syndecan-1.
 8. The antibody of claim 6, wherein the heparan sulfate proteoglycan is syndecan-2.
 9. The antibody of claim 6, wherein the heparan sulfate proteoglycan is syndecan-3.
 10. The antibody of claim 6, wherein the heparan sulfate proteoglycan is syndecan-4.
 11. The antibody of claim 6, wherein the heparan sulfate proteoglycan is a glypican, a betaglycan, CD44, perlecan, agrin, or collagen XVIII.
 12. The antibody of claim 1 that selectively binds to a biological sample comprising cancer cells when compared to a control sample.
 13. The antibody of claim 12, wherein the control sample and the biological sample comprise cells of the same tissue origin.
 14. The antibody of claim 12 or 13, wherein binding of the antibody to the biological sample is at least 2-fold greater than the binding of the antibody to the control sample.
 15. The antibody of claim 12 or 13, wherein binding of the antibody to the biological sample is at least 5-fold greater than the binding of the antibody to the control sample.
 16. The antibody of any one of claims 12-15, wherein the cancer cells comprise melanoma cells.
 17. The antibody of claim 1 that competes with antibody 15F7 characterized by a mature heavy chain variable region of SEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17, or antibody 6C12 characterized by a mature heavy chain variable region of SEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27, or antibody 13G10 characterized by a mature heavy chain variable region of SEQ ID NOS:36 or 37 and mature light chain variable region of SEQ ID NO:38.
 18. The antibody of claim 1 that binds to the same epitope on laminin α4 as 15F7, 6C12, or 13G10.
 19. The antibody of claim 1 comprising three light chain CDRs and three heavy chain CDRs, wherein each CDR has at least 90% sequence identity to a corresponding CDR from the heavy and light chain variable regions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively).
 20. The antibody of claim 1 comprising three heavy chain CDRs and three light chain CDRs of 15F7, 6C12, or 13G10.
 21. The antibody of any one of claim 1-20 or 88-89 that is monoclonal.
 22. The antibody of any one of claim 1-21 or 88-89 that is chimeric, humanized, veneered, or human.
 23. The antibody of any one of claim 1-22 or 88-89 that has human IgG1 kappa isotype.
 24. A humanized or chimeric 15F7 antibody that specifically binds to laminin α4, wherein 15F7 is a mouse antibody characterized by a mature heavy chain variable region of SEQ ID NO:16 and a mature light chain variable region of SEQ ID NO:17.
 25. The humanized antibody of claim 24 comprising a humanized heavy chain comprising three CDRs of the 15F7 heavy chain variable region (SEQ ID NO:16) and a humanized light chain comprising three CDRs of the 15F7 light chain variable region (SEQ ID NO:17).
 26. The humanized antibody of claim 24 comprising a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:58 and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:59.
 27. The humanized antibody of claim 26 comprising a humanized heavy chain comprising three CDRs of the 15F7 heavy chain variable region (SEQ ID NO:16) and a humanized light chain comprising three CDRs of the 15F7 light chain variable region (SEQ ID NO:17).
 28. The humanized antibody of claim 27, provided at least one of positions L42, L49, and L69 is occupied by N, S, and K, respectively, and at least one of positions H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 is occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively.
 29. The humanized antibody of claim 28, provided positions L42, L49, and L69 are occupied by N, S, and K, respectively, and H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 are occupied by E, L, Y, T, K, R, I, K, A, S, R, and F, respectively.
 30. The humanized antibody of any one of claims 27-29, provided at least one of positions L8, L45, and L80 is occupied by S, R, and T, respectively.
 31. The humanized antibody of any one of claims 27-29, provided at least one of positions H41 and H69 is occupied by A and L, respectively.
 32. The humanized antibody of claim 30 or 90, provided positions L8, L45, and L80 are occupied by S, R, and T, respectively.
 33. The humanized antibody of claim 31, provided positions H41 and H69 are occupied by A and L, respectively.
 34. The humanized antibody of claim 26, comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:58 and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:59.
 35. The humanized antibody of claim 26, wherein the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:57 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:59.
 36. The humanized antibody of claim 26, wherein the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:57 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:60.
 37. The humanized antibody of claim 26, wherein the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:58 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:59.
 38. The humanized antibody of claim 26, wherein the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:58 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:60.
 39. The antibody of any one of claim 1-38 or 88-91 that is an intact antibody.
 40. The antibody of any one of claim 1-38 or 88-91 that is a single-chain antibody, Fab, or Fab′2 fragment.
 41. The humanized antibody of any one of claim 24-40 or 90-91, wherein the mature light chain variable region is fused to a light chain constant region and the mature heavy chain variable region is fused to a heavy chain constant region.
 42. The humanized antibody of claim 41, wherein the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fcγ receptor relative to the natural human heavy chain constant region.
 43. The humanized antibody of claim 41 or 42, wherein the heavy chain constant region is of IgG1 isotype.
 44. The humanized antibody of claim 41, wherein the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO:61 and/or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO:62.
 45. The humanized antibody of any one of claim 24-44 or 90-92, provided any differences in CDRs of the mature heavy chain variable region and mature light chain variable region from SEQ ID NOS:16 and 17, respectively reside in positions H60-H65.
 46. The antibody of any one of claim 1-45 or 88-92, wherein the antibody is conjugated to a therapeutic or cytotoxic agent.
 47. The antibody of claim 46, wherein the cytotoxic agent is saporin.
 48. The antibody of claim 46, wherein the cytotoxic agent is a radioisotope.
 49. A pharmaceutical composition comprising the antibody of any one of claim 1-48 or 88-92 and a pharmaceutically acceptable carrier.
 50. A nucleic acid encoding the heavy and/or light chain(s) of an antibody as described in any one of claim 1-45 or 88-92.
 51. The nucleic acid of claim 50 having a sequence comprising any one of SEQ ID NOS:63-64, 67-68, 71-73, or 77-80.
 52. A recombinant expression vector comprising a nucleic acid of any one of claim 50, 51, or
 93. 53. A host cell transformed with the recombinant expression vector of claim
 52. 54. A method of humanizing an antibody, the method comprising: (a) determining the sequences of the heavy and light chain variable regions of a mouse antibody; (b) synthesizing a nucleic acid encoding a humanized heavy chain comprising CDRs of the mouse antibody heavy chain and a nucleic acid encoding a humanized light chain comprising CDRs of the mouse antibody light chain; (c) expressing the nucleic acids in a host cell to produce a humanized antibody; wherein the mouse antibody is 15F7, 6C12, or 13G10.
 55. A method of producing a humanized, chimeric, or veneered antibody, the method comprising: (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the antibody, so that the cells secrete the antibody; and (b) purifying the antibody from cell culture media; wherein the antibody is an antibody is a humanized, chimeric, or veneered form of 15F7, 6C12, or 13G10.
 56. A method of producing a cell line producing a humanized, chimeric, or veneered antibody, the method comprising: (a) introducing a vector encoding heavy and light chains of an antibody and a selectable marker into cells; (b) propagating the cells under conditions to select for cells having increased copy number of the vector; (c) isolating single cells from the selected cells; and (d) banking cells cloned from a single cell selected based on yield of antibody; wherein the antibody is a humanized, chimeric, or veneered form of 15F7, 6C12, or 13G10.
 57. The method of claim 56, further comprising propagating the cells under selective conditions and screening for cell lines naturally expressing and secreting at least 100 mg/L/10⁶ cells/24 h.
 58. A method of treating or effecting prophylaxis of a cancer in a patient having or at risk for the cancer, the method comprising administering to the patient an effective regime of the antibody of any one of claim 1-48 or 88-92.
 59. The method of claim 58, wherein the patient has a cancer, and the cancer is melanoma, breast cancer, lung cancer, or colorectal cancer.
 60. A method of inhibiting cell adhesion in a biological sample, the method comprising contacting the biological sample with an effective amount of the antibody of any one of claim 1-48 or 88-92.
 61. The method of claim 60, wherein the cell adhesion is mediated by the LG4-5 modules of the G domain of laminin α4.
 62. The method of claim 60, wherein the biological sample comprises cancer cells.
 63. A method for detecting the presence of a cancer in a biological sample, the method comprising: (a) contacting the biological sample with the antibody of any one of claim 1-48 or 88-92; (b) detecting binding of the antibody to the biological sample; (c) contacting a control sample with the antibody of any one of claim 1-48 or 88-92; (d) detecting binding of the antibody to the control sample; and (e) comparing binding of the antibody to the biological sample with binding of the antibody to the control sample, whereby increased binding of the antibody to the biological sample compared to the control sample indicates the presence of cancer in the biological sample.
 64. The method of claim 63, wherein the control sample and the biological sample comprise cells of the same tissue origin.
 65. The method of claim 63 or 64, wherein binding of the antibody to the biological sample is at least 2-fold greater than the binding of the antibody to the control sample.
 66. The method of claim 63 or 64, wherein binding of the antibody to the biological sample is at least 5-fold greater than the binding of the antibody to the control sample.
 67. The method of any one of claims 63-66, wherein the cancer is melanoma.
 68. A method of evaluating the efficacy of a therapeutic agent in a patient diagnosed with a cancer, the method comprising: (a) contacting a first biological sample from the patient, obtained prior to treatment with the therapeutic agent, with the antibody of any one of claim 1-48 or 88-92; (b) detecting binding of the antibody to the first biological sample; (c) contacting a second biological sample from the patient, obtained following treatment with the therapeutic agent, with the antibody of any one of claim 1-48 or 88-92; (d) detecting binding of the antibody to the second biological sample; (e) comparing binding of the antibody to the first biological sample with binding of the antibody to the second biological sample, whereby decreased binding of the antibody to the second biological sample compared to the first biological sample indicates that the therapeutic agent is effective in treating the cancer in the patient.
 69. A method of inhibiting binding of laminin α4 to a heparan sulfate proteoglycan in a biological sample, the method comprising contacting the biological sample with the antibody of any one of claim 1-48 or 88-92.
 70. The method of claim 69, wherein the heparan sulfate proteoglycan is syndecan-1.
 71. The method of claim 69, wherein the heparan sulfate proteoglycan is syndecan-2.
 72. The method of claim 69, wherein the heparan sulfate proteoglycan is syndecan-3.
 73. The method of claim 69, wherein the heparan sulfate proteoglycan is syndecan-4.
 74. The method of claim 69, wherein the heparan sulfate proteoglycan is a glypican, a betaglycan, CD44, perlecan, agrin, or collagen XVIII.
 75. A method of treating or effecting prophylaxis of a disease in which the LG4-5 modules of the G domain of laminin α4 contribute to progression of the disease, the method comprising administering to a patient having or at risk of the disease an effective regime of the antibody of any one of claim 1-48 or 88-92.
 76. The method of claim 75, wherein the disease is a cancer.
 77. The method of claim 76, wherein the cancer is melanoma, breast cancer, lung cancer, or colorectal cancer.
 78. The method of claim 75, wherein the LG4-5 modules of the G domain of laminin α4 contribute to progression of the disease by means of interaction with a heparan sulfate proteoglycan.
 79. The method of claim 78, wherein the heparan sulfate proteoglycan is syndecan-1.
 80. The method of claim 78, wherein the heparan sulfate proteoglycan is syndecan-2.
 81. The method of claim 78, wherein the heparan sulfate proteoglycan is syndecan-3.
 82. The method of claim 78, wherein the heparan sulfate proteoglycan is syndecan-4.
 83. The method of claim 75, wherein the disease is psoriasis, sarcoidosis, multiple sclerosis, or psoriatric arthritis.
 84. A method of treating or effecting prophylaxis of an autoimmune disease in a patient having or at risk for the autoimmune disease, the method comprising administering to the patient an effective regime of the antibody of any one of claim 1-48 or 88-92.
 85. The method of claim 84, wherein the disease is diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, celiac disease, psoriasis, psoriatic arthritis, sarcoidosis, ankylosing spondylitis, Sjogren's syndrome, or uveitis.
 86. A method of inhibiting angiogenesis in a patient, the method comprising administering to the patient an effective regime of the antibody of any one of claim 1-48 or 88-92.
 87. The method of claim 86, wherein the patient has a cancer.
 88. The antibody of claim 1 that competes with antibody 15F7 characterized by a mature heavy chain variable region of SEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17, or antibody 6C12 characterized by a mature heavy chain variable region of SEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27 or 94, or antibody 13G10 characterized by a mature heavy chain variable region of SEQ ID NOS:36 or 37 and mature light chain variable region of SEQ ID NO:38.
 89. The antibody of claim 1 comprising three light chain CDRs and three heavy chain CDRs, wherein each CDR has at least 90% sequence identity to a corresponding CDR from the heavy and light chain variable regions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27/94, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively).
 90. The humanized antibody of any one of claims 27-29, provided at least one of positions L8, L45, L80, and L104 is occupied by S, R, T, and V, respectively.
 91. The humanized antibody of claim 90, provided position L104 is occupied by V.
 92. The humanized antibody of claim 41, wherein the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO:61, 89, or 101 and/or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO:62 or
 90. 93. The nucleic acid of claim 50 having a sequence comprising any one of SEQ ID NOS:63-64, 67-68, 71-73, 77-80, 92-93, 99-100, or
 102. 94. A method of treating or effecting prophylaxis of obesity or an obesity-related disease in a patient having or at risk for obesity or the obesity-related disease, the method comprising administering to the patient an effective regime of the antibody of any one of claim 1-48 or 88-92.
 95. The method of claim 79, wherein the obesity-related disease is non-alcoholic steatohepatitis (NASH), Prader-Willi syndrome, craniopharyngioma, Bardet-Biedl syndrome, Cohen syndrome, or MOMO syndrome. 